DNA transfection system for the generation of infectious influenza virus

ABSTRACT

The present invention is based on the development of a dual promoter system (preferably a RNA pol I-pol II system) for the efficient intracellular synthesis of viral RNA. The resultant minimal plasmid-based system may be used to synthesize any RNA virus, preferably viruses with a negative single stranded RNA genome. The viral product of the system is produced when the plasmids of the system are introduced into a suitable host cell. One application of the system is production of attenuated, reassortant influenza viruses for use as antigens in vaccines. The reassortant viruses generated by cotransfection of plasmids may comprise genes encoding the surface glycoproteins hemagglutinin and neuraminidase from an influenza virus currently infecting the population and the internal genes from an attenuated influenza virus. An advantageous property of the present invention is its versatility; the system may be quickly and easily adapted to synthesize an attenuated version of any RNA virus. Attenuated or inactivated RNA viruses produced by the present invention may be administered to a patient in need of vaccination by any of several routes including intranasally or intramuscularly.

This application claims the benefit of U.S. Provisional Application No.60/200,679, filed Apr. 28, 2000 which is herein incorporated byreference in its entirety.

The studies that led to this invention were supported by Public HealthResearch Grants AI95357, AI29680, AI08831, AI29559 and AI29680 from theNational Institute of Allergy and Infectious Diseases. Accordingly, theUnited States Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the development of a minimumplasmid-based system for the generation of infectious RNA viruses,preferably influenza viruses, from cloned DNA. In particular, thismulti-plasmid pol I-pol II system facilitates the generation of bothrecombinant and reassortment viruses. In preferred embodiments, theinvention comprises an eight plasmid pol I-pol II system for generationof influenza viruses. It also has applicability in the recovery of otherRNA viruses entirely from cloned cDNA.

BACKGROUND OF THE INVENTION Life Cycle of RNA Viruses

The genomes of RNA viruses have different configurations, includingunimolecular or segmented; single stranded of (+) or (−) polarity ordouble stranded. However, two essential, common requirements are sharedbetween the viruses: (1) the genomic RNAs must be efficiently copiedinto a form which can be effectively used for assembly into progenyvirus particles and (2) mRNAs which can be efficiently translated intoviral proteins must be synthesized. Generally, RNA viruses (exceptretroviruses) encode and/or carry an RNA-dependent RNA polymerase tocatalyze synthesis of new genomic RNA (for assembly into progeny) andmRNAs (for translation into viral proteins). Since eukaryotic host cellstypically do not contain machinery for replicating an RNA template orfor translating polypeptides from a negative stranded or double strandedRNA template, viruses comprising these nucleic acids in their genomesmust carry an RNA polymerase protein in the viral particle. For thisreason, deproteinized RNA molecules of negative stranded and doublestrand RNA viruses (lacking an associated RNA polymerase) arenoninfectious. In contrast, deproteinized RNA from the genome of apositive stranded RNA virus is, typically, infectious because encodedviral proteins are translatable by host cellular machinery.

Genomic viral RNA must be packaged into viral particles in order for thevirus to be transmitted. Some RNA virus capsids are enveloped by lipidmembranes from the infected host cells and others have an outer viralprotein shell without a lipid bilayer. Despite these differences betweenviral capsids, the process by which progeny viral particles areassembled and the protein/protein interactions which occur duringassembly are similar. Viral proteins are generally classified asstructural and nonstructural proteins. In general, nonstructuralproteins are involved in genomic replication, regulation oftranscription and packaging. The structural proteins generally performthree types of functions including: (1) binding to genomic RNA (i.e,nucleocapsid protein for influenza A virus), (2) bridging betweenpackaged RNA and outer proteins (i.e., matrix protein) and (3) buildingan outer viral layer (i.e., surface proteins such as hemagglutinin). Theassembly into virus particles ensures the effective transmission of theRNA genome from one host cell to another within a single host or amongdifferent host organisms.

Influenza Virus

Influenza A virus, an Orthomyxoviridae, is a negative-sense RNA viruswith a segmented genome. The genomic RNAs contain one or more openreading frames flanked by noncoding sequences at the 5′ and 3′ ends(Desselberger et al., Gene 1980, 8:315). Viral RNAs are associated withviral nucleoprotein (NP) and polymerase proteins (PB1, PB2 and PA) invirions and in infected cells to form ribonucleoprotein (RNP) complexes(Hsu et al., Proc. Natl. Acad. Sci. USA 1987, 84:8140). Its geneticcomposition allows this virus to evolve by reassortment of gene segmentsfrom different strains; this reassortment creates new variants for whicha newly infected organism has no anamnestic immune response. Of the 15hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenzacirculating in aquatic birds, three, H1N1, H2N2, and H3N2 subtypes areknown to have caused pandemics in humans (Webster et al., Microbiol.Rev. 1992, 56:152). There is evidence that pigs can serve as anintermediate host (“mixing vessel”) for the generation of new strainsthat are pathogenic in humans (Scholtissek et al., Virology 1985,147:287). The H5N1 influenza A outbreak in Hong Kong in 1997 showed thathighly pathogenic influenza A viruses can also be transmitted directlyfrom avian species to humans (Claas et al., Lancet 1998, 351:472; Suarezet al., J. Virol. 1998, 72:6678; Subbarao et al., Science 1998, 279:393;Shortridge, Vaccine 1999, 17 (Suppl. 1): S26-S29). The potential ofinfluenza A viruses to generate new pathogenic strains from a vastnumber of circulating strains in the natural reservoir indicates thatdisease control requires monitoring these viruses and developingimproved antiviral therapies and vaccines. The speed with which newstrains develop demands vigilance in this monitoring effort, andstretches the capacity of current technology to produce sufficientquantities of vaccine against a newly identified pathogenic strain toprevent an epidemic or pandemic.

For influenza A virus, reverse-genetics systems have allowed themanipulation of the viral genome (Palese et al., Proc. Natl. Acad. Sci.USA 1996, 93:11354; Neumann and Kawaoka; Adv. Virus Res. 1999, 53:265).Unlike positive-strand viruses (i.e., poliovirus), the negative-senseviral RNAs (vRNAs) of influenza A viruses are not infectious. Only vRNAmolecules encapsidated with the four viral polymerase complex proteins(PB1, PB2, PA, NP) are able to initiate a viral replication andtranscription cycle. After the ribonucleoproteins (RNPs) penetrate thecell nucleus, the associated proteins begin to transcribe the (−) vRNAsinto mRNAs and positive sense complementary RNAs (+) cRNAs. These cRNAsserve as templates for the synthesis of vRNAs. The firstreverse-genetics system to be developed for influenza A virus was theRNA-transfection method (Luytjes et al., Cell 1989, 59:1107; Enami etal., Proc. Natl. Acad. Sci. USA 1990, 87:3802). After in vitrotranscription of virus-like vRNA by the T7 RNA polymerase andreconstitution of viral ribonucleoprotein (vRNA) molecules, geneticallyaltered RNP segments were introduced into eukaryotic cells bytransfection. Infection with influenza helper virus resulted in thegeneration of viruses possessing a gene derived from cloned cDNA.However, the presence of helper virus in RNA and DNA transfectionmethods severely limits the practical value of these methods since astrong selection system is required to eliminate helper virus.

The establishment of the RNA polymerase I (pol I)-driven synthesis ofvRNA molecules in vivo allowed the intracellular production of RNAcomplexes (Neumann and Hobom, Virology 1994, 202:477). In this system,virus-like cDNA was inserted between the pol I promoter and terminatorsequences (Zobel et al., Nucl. Acids Res. 1993, 21:3607). Unlike themRNA transcripts synthesized by RNA polymerase II (pol II), polI-generated RNAs lack both a 5′ cap and a 3′ poly (A) tail. FunctionalvRNP molecules could be generated either by infection with helper virusor by cotransfection of protein expression plasmids encoding PB1, PB2,PA, or NP (Neumann and Hobom, supra; Flick et al., RNA 1996, 2:1046;Pleschka et al., J. Virol. 1996, 70:4188; Zhou et al., Virology 1998,246:83).

Recent studies demonstrated that the plasmid-driven expression of alleight vRNAs from a pol I promoter and the coexpression of the polymerasecomplex proteins result in the formation of infectious influenza A virus(Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al.,J. Virol. 1999, 73:9679). Because the generation of influenza A virusdriven entirely from plasmids requires no infection with helper virus,no selection system is needed; therefore, all gene segments can bemanipulated without technical limitations. In the system developed byNeumann et al. (supra), the eight cDNAs were inserted between a humanpol I promoter sequence (407 bp) and a murine terminator sequence (174bp). Expression of the four RNP-complex proteins was driven by the humancytomegalovirus promoter. Transfection of 12 plasmids into 10⁶ 293Tcells resulted in virus recovery of more than 10³ pfu; this efficiencycould be increased to 5×10⁷ pfu after the transfection of 17 plasmids.Fodor et al. (supra) developed a system in which the eight cDNAs wereinserted between a human pol I promoter sequence (250 bp) and a genomicribozyme sequence of hepatitis delta virus to ensure the precise 3′ endof the vRNA. For the expression of the polymerase complex genes,plasmids containing the adenovirus type 2 major late promoter were used.After transfection of the 12 expression plasmids into Vero cells, onlyone or two infectious viral particles were rescued from 10⁶ transfectedcells.

However, the helper-virus-free system described by Neumann et al.(supra), which contains the pol I and pol II promoters with theinfluenza virus cDNAs on different plasmids, requires the constructionand cotransfection of at least 12 plasmids for virus recovery, and 17plasmids for efficient virus recovery. Transfection of cells with thismany number of plasmids may limit the use of this system to cell lineswhich have a high transfection efficiency. To be able to rescue virusfrom different cell types may increase the virus yield by enhancing thereplication of influenza A virus in these cells and increase the rangeof cells suitable for the production of vaccines (Govorkova et al., J.Virol. 1996, 70:5519).

Thus, there is a need in the art for more efficient generation ofrecombinant influenza viruses. Moreover, there is a further need in theart for efficient generation of reassortment viruses for vaccineproduction in response to a newly identified virus strain. The presentinvention addresses these and other needs in the art by providingsystems in which synthesis of both viral genomic negative strand RNAsegments (vRNA) and viral mRNA occurs from one template, therebyminimizing the number of plasmids required for virus generation andpermitting efficient and predictable reassortment.

Reoviridae Viruses

Viruses from the family Reoviridae, including viruses of the genusRotavirus, comprise a double stranded, segmented RNA genome. Humanrotavirus is the most common viral agent of severe childhood diarrhea inthe United States, causing about 50,000 hospitalizations and 20 to 50deaths per year at an estimated annual cost of more than $1 billion. Indeveloping countries, it is estimated that rotavirus is responsible forone-third of all diarrhea-associated hospitalizations and causeapproximately 850,000 deaths annually.

A dual system of reporting rotavirus serotypes exists due to theneutralizing response evoked by two viral proteins (VP), VP7 and VP4.The VP7 serotypes are designated G types, and those derived from VP4 aredescribed as P types. To date, at least 10 G serotypes and at least 7 Pserotypes are found in humans. Since VP4 and VP7 genes segregateseparately, new rotaviruses are generated by reassortment. In the UnitedStates, the serotypes P1 to P4 and G1 to G4 are most frequent; othercombinations were reported in countries like India and Egypt. The firstlicensed human rotavirus vaccine, the rhesus rotavirus vaccine, wasformulated to produce serotype-specific protection against the fourcommon serotypes, G1 to G4. However, this vaccine was withdrawn becauseof an association between vaccination and increased rates ofintussusception among vaccine recipients. Thus, there is a need forproducing a rotavirus vaccine representing all G and P subtypes whichhas no unwanted side effects. The current invention provides vectors,(preferably plasmids), methods and host cells which can be employed forgenerating rotaviruses entirely from cloned cDNA.

Thirteen primary gene products have been defined. To minimize confusionand to facilitate the comparison with proteins with similar functionsfrom other genera of the Reoviridae, the following nomenclature has beenemployed: according to their migration in SDS-PAGE analysis, startingwith the largest protein, the structural proteins have been given theprefix “VP” and nonstructural proteins the prefix “NSP” and the functionof each protein is given in brackets. For example, the abbreviationVP1(Pol) indicates that the largest protein in virus particles is theRNA-dependent RNA polymerase. The seven structural proteins assembleinto viral particles which comprise three layers of structure: (1) Theinner viral core containing the dsRNA genome has three proteinsassociated with it, two of which (VP1(Pol) and VP3 (Cap)) are directlyassociated with the genome whereas the third (VP2(T2)) makes up the coreshell, (2) the middle protein shell of the virion is made up of 780VP6(T13) molecules arranged in 260 trimeric units and (3) VP4 and VP7make up the outer shell. The spike protein VP4 contains a trypsincleavage site that is important for cleavage into VP5 and VP8, and thiscleavage enhances infectivity. Two forms of VP7, derived from differentinframe reading frames, VP7(1) and VP7(2), are sought to be incorporatedinto virions.

Much less is known about the functions of the six nonstructuralproteins. Similar to other RNA viruses, it is anticipated that thenonstructural proteins play important roles in virus replication,transcription, translation of viral RNAs and packaging. Indeed, based onthe analyses of temperature sensitive viruses in segment 8, it ishypothesized that NSP2(ViP) has a direct role in virus replication. NSP3is believed to bind to conserved sequences at the 3′-end of viral mRNAsand to the cellular cap binding protein eIF4G thereby specificallyupregulating translation of rotavirus mRNAs which have 5′-cap structuresbut no 3′-polyA-tails. NSP1 appears to be nonessential, but it probablyplays an active role in rotavirus replication in cell culture. NSP4 isbelieved to be involved in virus morphogenesis. The two nonstructuralproteins, NSP5 and NSP6, are encoded by two different reading framesfrom segment 11, but their function in the viral life cycle is notknown.

The replication cycle is completed in 10-12 hours at 37° C. Current datasuggest that viruses can enter cells through receptor-mediatedendocytosis but there may be an alternative mechanism for cell entry.After entering the host cell, the outer virus shell releases thetranscriptionally active double-shelled particle into the cytoplasm ofthe infected cell. Virion-associated enzymes produce 5′-capped,nonpolyadenylated mRNAs, which are full-length transcripts from theminus strand of each of the virion genome segments. The viral mRNAsderived from each segment serve two functions: first, they aretranslated to generate the viral proteins encoded by the segment andsecond, viral mRNAs are also the templates for genome replication.Genome segment assembly takes place by selection of the different viralmRNAs required to form precore RI. Assembly of the 11 mRNAs is followedby minus strand synthesis, which occurs in ‘core-RI’ and VP6(T13)-RI,which are present in the ‘viroplasms’ found in the cytoplasm of infectedcell. The next steps in morphogenesis of progeny virions are unique torotaviruses and involve double-layered particle budding into theendoplasmic reticulum in a process that involves NSP4. This results inthe particle transiently acquiring an envelope that is lost during thefinal maturation steps when the outer virion shell of VP4 and VP7 isadded.

A segmented genome, a highly ordered genomic structure and a complexreplication cycle present major challenges for the development of areverse genetic system for generation of rotaviruses. However, thepresent invention may be used for simple and convenient generation ofrotavirus.

Influenza Vaccines

The influenza vaccines currently licensed by public health authoritiesfor use in the United States and Europe are inactivated influenzavaccines. The viruses presenting epidemiologically important influenza Aand influenza B strains are grown in embryonated hens' eggs and thevirus particles are subsequently purified and inactivated by chemicalmeans. Each year the WHO selects subtypes which most likely willcirculate: currently two strains for influenza A (H1N1) and (H3N2), anda B strain.

For the production of a safe and effective vaccine it is important thatthe selected vaccine strains are closely related to the circulatingstrains, thereby ensuring that the antibodies in the vaccinatedpopulation are able to neutralize the antigenetically similar virus.However, not all viruses found to be closely related are suitable forvaccine production because they grow poorly in eggs. Therefore, it isdesirable to attempt to generate a high growth reassortment virus tocombine the high virus yield of a laboratory strain (A/PR/8/34) (H1N1)with the antigenic characteristics of the anticipated pathogenic strain.Unfortunately, coinfection with two influenza viruses containing eightsegments results in the generation of theoretically 2⁸=256 differentprogeny viruses. To obtain a high growth virus with the requiredglycoprotein antigens, a selection method is needed to eliminate thecorresponding gene segments from the parental high growth laboratorystrain. The selection procedure to obtain the virus with the appropriateglycoproteins and the verification of the gene constellation is acumbersome and time consuming task. Although the RNP-transfection system(Luytjes et al., Cell 1989, 59:1107) reduces the possible number ofprogeny virus, a good selection method is still required.

Live attenuated influenza virus vaccines administered intranasallyinduce local, mucosal, cell-mediated and humoral immunity. Cold-adapted(ca) reassortment (CR) viruses containing the six internal genes oflive, attenuated influenza A/Ann Arbor/6/60 (H2N2) or B/Ann Arbor/1/66,and the haemagglutinin (HA) and neuraminidase (NA) of contemporarywild-type influenza viruses appear to be reliably attenuated. Thisvaccine appears to be efficacious in children and young adults. However,it may be too attenuated to stimulate an ideal immune response inelderly people, the major group of the 20,000-40,000 individuals in theUSA dying each year as a result of influenza infection. Although thesequences of the internal genes of the ca viruses have been reported,the contribution of each segment to the attenuated phenotype is stillnot well defined. This information can be acquired only by thesequential introduction of specific, defined attenuating mutations intoa virus. Although the RNP-transfection method allows the introduction ofmutation into the genome of influenza, the need for a selection systemand the technical difficulties. of reconstituting viral RNPs in vitrolimits the use for the manipulation of the internal genes.

Thus, there is a need in the art for development of recombinantinfluenza vaccines that avoid the use of helper virus, grow well inculture (eggs or cell culture), reliably permit development ofreassortment viruses that can be propagated for new vaccine development,and provide for systematic mutation to develop live attenuated virusstrains for intranasal vaccination. The present invention addressesthese and other needs in the art.

SUMMARY OF THE INVENTION

The present invention advantageously provides an expression plasmidcomprising an RNA polymerase I (pol I) promoter and pol I terminatorsequences, which are inserted between an RNA polymerase II (pol II)promoter and a polyadenylation signal. The expression plasmid is termedherein a pol I-pol II system, a dual promoter expression system or dualpromoter expression plasmid. Such a plasmid optimally contains an RNAvirus viral gene segment inserted between the pol I promoter and thetermination signal. Preferably, the RNA virus is an influenza virus(e.g., an influenza A or influenza B virus).

The invention comprises two plasmid based systems for generatinginfectious RNA viruses from cloned genes or cDNA. In one system(bidirectional system), the gene or cDNA is located between an upstreampol II promoter and a downstream pol I promoter. Transcription of thegene or cDNA from the pol II promoter produces capped positive-senseviral mRNA and transcription from the pol I promoter producesnegative-sense, uncapped vRNA. In the other system (unidirectionalsystem), the gene or cDNA is located downstream of a pol I and a pol IIpromoter. The pol II promoter produces capped positive-sense viral mRNAand the pol I promoter produces uncapped positive-sense viral cRNA.

A minimum plasmid-based system of the invention permits generation ofinfectious RNA viruses from cloned viral cDNA. Such a system comprises aset of plasmids wherein each plasmid comprises one autonomous viralgenomic segment of the RNA virus. In each plasmid, the viral cDNA,corresponding to the autonomous viral genomic segment, is insertedbetween an RNA polymerase I (pol I) promoter and terminator sequences,thereby resulting in expression of vRNA, which are in turn insertedbetween a RNA polymerase II (pol II) promoter and a polyadenylationsignal, thereby resulting in expression of viral mRNA. Thus, this systememploys the bidirectional plasmid technology, and permits efficientreassortment to produce RNA viruses corresponding to the currentpathogenic strains in circulation, e.g., in terms of the influenza NAand HA genes, in a background strain well adapted to grow in cellculture or from an attenuated strain, or both. Preferably the virus isan influenza A virus or an influenza B virus.

The invention provides host cells comprising the plasmid-based systemfor the generation of infectious virions, and methods for producing RNAvirus virions, which methods comprise culturing the host cell underconditions that permit production of viral proteins and vRNA.

The plasmid-based system, host cells, and method for producing virionsare particularly suited to preparing an RNA virus-specific vaccine. Suchmethods comprise purifying virions. Purified virions can be inactivatedor may be attenuated. Vaccines of the invention can be used forvaccinating against an RNA virus infection. For example, a protectivedose of a vaccine comprising inactivated virions can be administered byintramuscular injection. Alternatively, a protective dose of a vaccinecomprising attenuated virions can be administered intranasally to asubject.

The invention further provides reassortment virus virions, and vaccinecompositions comprising such virions, including inactivated andattenuated virions.

In another advantageous embodiment, the invention provides a method forgenerating an attenuated RNA virus. This method comprises mutating oneor more viral genes in the plasmid-based system, and then determiningwhether infectious RNA viruses produced by the system are attenuated.Such attenuated viruses can be used to develop intranasal vaccines,including intranasal vaccines with enhanced potency to elicit protectiveimmunity in aged or other populations who are non-responsive to currentattenuated vaccines.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the pol I-pol II transcriptionsystem for synthesis of vRNA and mRNA; The cDNA of each of the eightinfluenza virus segments is inserted between the pol I promoter (P_(Ih))and the pol I terminator (t_(I)). This pol I transcription unit isflanked by the pol II promoter (p_(II) CMV) of the human cytomegalovirusand the polyadenylation signal (a_(II) BGH) of the gene encoding bovinegrowth hormone. After transfection of the eight expression plasmids, twotypes of molecules are synthesized. From the human pol I promoter,negative-sense vRNA is synthesized by cellular pol I. The synthesizedvRNA contains the noncoding region (NCR) at the 5′ and 3′ ends.Transcription by pol II yields mRNAs with 5′ cap structures and 3′ polyA tails; these mRNAs are translated into viral proteins. The ATG of theviral cDNA is the first ATG downstream of the pol II transcription startsite.

FIG. 2. The eight plasmid pol I-pol II system for the generation ofinfluenza A virus. Eight expression plasmids containing the eight viralcDNAs inserted between the human pol I promoter and the pol II promoter(see FIG. 1) are transfected into eukaryotic cells. Because each plasmidcontains two different promoters, both cellular pol I and pol II willtranscribe the plasmid template, presumably in different nuclearcompartments, which results in the synthesis of viral mRNAs and vRNAs.After synthesis of the viral polymerase complex proteins (PB1, PB2, PA,NP), the viral replication cycle is initiated. Ultimately, the assemblyof all viral molecules directly (pol II transcription) or indirectly(pol I transcription and viral replication) derived from the cellulartranscription and translation machinery results in the interaction ofall synthesized molecules (vRNPs and the structural proteins HA, NA, M1,M2, NS2/NEP) to generate infectious influenza A virus.

FIGS. 3A and 3B. Schematic representation of the method developed forthe construction and transfection of the eight expression plasmids torecover A/Teal/HK/W312/97 (H6N1). A. Viral RNA was extracted from virusparticles. RT-PCR was performed with primers containing segment-specificnucleotides and sequences for the type IIs restriction endonucleasesBsmBI or BsaI. The eight viral PCR fragments were digested with BsmBI orBsaI and inserted into pHW2000 (linearized with BsmBI). This insertionresulted in eight expression constructs where the viral cDNAs areprecisely fused to the pol I promoter and terminator (the viral terminalsequences AGC . . . ACT are shown for the PB2 segment in the blackrectangles). B. The eight expression plasmids with a pol I promoter anda pol II promoter contain one copy of each of the viral cDNAs of theeight segments. The open reading frames for the 10 viral proteins, areflanked by the segment-specific noncoding regions (gray boxes). Becausethe used human pol I promoter shows high activity only in cell linesderived from humans or related species, human 293T cells were coculturedtogether with the standard cell line used for influenza A (MDCK-cells).Viruses produced in the 293T cells after transfection can then infectMDCK cells and replicate.

FIGS. 4A and 4B. Characterization of the recovered viruses by RT-PCR. A.RNA was extracted from virus particles after two passages of thesupernatant of transfected cells (see Tables 1 and 2) on MDCK cells.RT-PCR was performed with primers specific for the NS gene segment andwith vRNA extracted from virions. The NS primers used were not strainspecific; thus, allowing the amplification of any influenza A NSsegment. The reaction products were subjected to electrophoresis on a 2%agarose gel. To ensure that the amplified DNA fragments were derivedfrom vRNA and not from plasmid DNA carried over from transfected cells,one reaction was performed without the addition of reverse transcriptase(RT) (−). Lanes 1 and 2, recombinant A/Teal/HK/W312/97 (Table 1); lanes3 and 4, M-reassortment (Table 2); Lanes 5 and 6, NS-reassortment (Table2); lanes 7 and 8, recombinant A/WSN/33 virus (Table 1); lanes 9 and 10,quadruple-reassortment (Table 2). B. NcoI digestion of the fragmentsshown in A. The identity of the NS fragments was also verified bysequence analysis of the amplified product (not shown).

FIG. 5. Unidirectional RNA pol I-pol II transcription system. In theunidirectional pol I-pol II transcription system, viral cDNA is insertedin the positive-sense orientation between a human pol I promoter(p_(I)h) and terminator sequence (t_(I)). This entire pol Itranscription unit is flanked by a pol II promoter (p_(IICMV): immediateearly promoter of the human cytomegalovirus) and the polyadenylationsite of the gene encoding bovine growth hormone (a_(IIbgh)). Aftertransfection, two types of RNA transcripts are expected to besynthesized. Positive-sense cRNA with a triphosphate group at its 5′ endsynthesized by pol I, and positive-sense mRNA synthesized by pol II witha 5′-cap structure and a poly(A) tail at its 3′ end. Both elements ofthe mRNA are required for efficient translation.

FIG. 6. The cloning vector pHW11 with a pol I and a pol II promoterarranged in tandem. The plasmid contains the 225-bp human RNA pol Ipromoter (p_(I)h) and the 33-bp murine terminator (t_(I)). The pol Ipromoter and terminator sequences are flanked by the RNA polymerase IIpromoter (p_(IICMV)) of the human cytomegalovirus and thepolyadenylation signal (a_(IIBGH)) of the gene encoding bovine growthhormone. For insertion of viral cDNA between the pol I promoter andterminator, two BsmBI restriction sites (indicated by underlining) wereintroduced. Digestion of the vector with BsmBI created a vector fragmentwith sticky but noncomplementary protruding ends. The design of thisvector allows the precise fusion of viral cDNA in the positive-senseorientation with respect to the pol I promoter and terminator sequence.For propagation in E. coli, the plasmid has an origin of replication(ori), and for selection in ampicillin-containing medium, the plasmidcontains a beta-lactamase gene (bla).

FIGS. 7A and 7B. Dual promoter system for the generation of infectiousRNA viruses. Since RNA viruses function as cellular parasites they mustoptimize strategies for using host cells for expression of their geneticinformation. All RNA viruses must synthesize mRNAs which are capable ofbeing translated into proteins. Generally, the synthesized proteins arerequired for replication, transcription and producing new progeny virusparticles. For efficient replication of the genomic RNAs,RNA-transcripts with exact 5′ and 3′ ends must be made.

The present system comprises an outer and an inner transcription unit.The inner transcription unit comprises a promoter (p (+RNA) or p(−RNA)),preferably a pol I promoter. The cDNAs of RNA viruses consist of one ormore open reading frames (ORF) which are flanked by non coding regions(NCR). Preferably, there are no sequences intervening between the viralcDNA and the promoter. The lack of intervening sequences is vitalbecause the 5′ and 3′ ends of genomic vRNA generally contain sequencesrecognized by viral proteins needed for transcription and replication;additional non-virus sequences typically impedes efficient recognitionand replication of the vRNA by viral proteins. The lack of interveningsequences allows the transcribed (−) strand RNA (A) or (+) strand RNA(B) to be used efficiently by viral polymerase proteins. The outertranscription unit has a promoter (p(mRNA)), preferably a pol IIpromoter which directs transcription of mRNA from the cDNA; the mRNAincludes 5′ sequences (e.g., methyl G caps) and 3′ sequences (e.g., polyA tails) which are required for translational initiation and productionof viral proteins. Since the process of translation is tolerant ofadditional sequences between the promoter, of the outer transcriptionunit, and the viral cDNA, the presence of intervening sequences from theinner transcription unit do not significantly impede translation of themRNA.

This system can be modified and improved for RNA viruses other thaninfluenza virus by using different promoters in the inner transcriptionunit (e.g., pol II, pol III, T3, SP6, T7 or any other promoter for aDNA-dependent RNA polymerase) and termination elements or ribozymes forthe intracellular synthesis of viral RNA with exact 5′ and 3′ ends(discussed infra). Hammerhead ribozymes or hepatitis delta virus (HDV)ribozyme can be employed for generatiion of viral RNA with exact ends(Schnell et al., EMBO J. 1994, 13:4195; Pleschka et al., J. Virol. 1996,70:4188; Herold, J. et al., J. Virol 2000, 74(14):6394-400).

The outer transcription unit may comprise a pol I or III promoter, a T7RNA polymerase promoter, a T3 RNA polymerase promoter, SP6 RNApolymerase promoter, or any other promoter for a DNA-dependent RNApolymerase. If the promoter in the outer transcription unit directssynthesis of a transcript which lacks a methyl G cap, an InternalRibosome Entry Site (IRES) may be placed at the 5′ end of the cDNAcoding sequence to facilitate translational initiation (discussedinfra).

It is noteworthy that the vector pHW2000 has a T7 promoter between theCMV-promoter and the termination site. Pol II transcripts aresynthesized in the nucleus, whereas T7-transcripts are synthesized inthe cytoplasm of cells expressing T7 RNA polymerase. Hence, transcriptsoriginating from more than one promoter of an outer transcription unitcan be produced resulting in different mRNAs. Thus, expression plasmidsderived from pHW2000 allow the rapid evaluation of whether the pol II orT7 promoter or the combination of both is optimal for mRNA synthesis ofpositive strand viruses which have an Internal Ribosome Entry Site(IRES).

FIG. 8. Dual promoter system for the generation of a (+) strand RNAviruses. The present invention may also be adapted to produce virusescomprising a positive strand, unimolecular genome, such as hepatitis Cvirus. In this embodiment, a cDNA comprising the hepatitis C virusgenome (approximately 9500 nucleotides) is inserted in a construct thatallows efficient transcription of the cDNA intracellularly into mRNA anda full length negative RNA (bidirectional approach) or mRNA and fulllength positive RNA (unidirectional approach). The cDNA consists of oneopen reading frame (ORF) which is flanked by the non-coding regions(NCR). In the figure, an expression plasmid containing the bidirectionalsystem is shown. The full length cDNA is inserted between a pol I(p_(I)) promoter and termination sequences (t_(I)) resulting in fulllength (−) strand RNA synthesis after transfection.

The inner transcription unit is flanked by an outer transcription unitwhich has a promoter (p(mRNA)) to drive mRNA synthesis. Preferably, thispromoter is a pol II promoter. However, if the synthesized RNA has aninternal ribosomal entry site (IRES), the pol II promoter may besubstituted by a pol I, pol III, SP6, T7 or T3 promoter (use of T3 or T7promoters requires that the T3 or T7 polymerase proteins be expressedeither by cotransfection of a plasmid encoding the polymerases or use ofa stable cell line expressing the polymerases). At the 3′-end of theouter transcription unit, either a poly A signal or an inserted poly Asequence is used to provide a polyA tail for the synthesized mRNA.

The resultant mRNA is translated into a large polyprotein precursor thatis cleaved co- and posttranslationally to yield individual structuraland nonstructural viral proteins.

The nonstructural proteins NS5a and NS5b, which are the RNA-dependentRNA polymerase proteins use the (−) RNA synthesized by the innertranscription unit as a template to initiate the viralreplication/transcription cycle. Thus, (+) RNA/mRNA is produced which isused for translation into protein. Ultimately infectious viruses aregenerated which contain (+) RNA together with viral structural proteins.

FIG. 9. Pol I-pol II system for the generation of human Parainfluenzavirus III. The present invention may be used to produce parainfluenzaIII virus which comprises a negative strand, unimolecular RNA genome.Viral cDNA could be inserted into the pol I-pol II system either in asense or an antisense orientation. In the figure the unidirectionalsystem is presented. A pol I promoter directs synthesis of cRNA and apol II promoter directs synthesis of mRNA. In this embodiment, a pol IIpromoter produces a polycistronic mRNA from which the first first openreading frame is efficiently translated into Nucleocapsid (NP) protein.This protein is required for replication. Plasmids encoding the L andP-protein, which are also essential for replication and transcription(but are not efficiently translated from the polycistronic mRNA), areprepared and are co-transfected on separate expression plasmids.Compared to the reverse genetics system developed by Durbin, A. P. etal., Virology 1997, 235(2):323-332, the pol I-pol II system has severaladvantages. By the expression of NP from the same cDNA, this minimumplasmid system requires the construction and transfection of only threeinstead of four plasmids to generate human Parainfluenzavirus IIIentirely from cloned cDNA. Unlike the reverse genetics systems based onthe in vivo transcription from the T7-promoter, the pol I-pol II systemis entirely driven by eucaryotic DNA dependent RNA polymerases found ineach cell. Moreover, the infection of vaccinia virus which drives theexpression of the T7 RNA polymerase requires the use of cells which arepermissive for vaccinia virus (HeLa cells or derivatives such as Hep-2cells) but not optimal for growth of human parainfluenza virus, thuslimiting the utility of this approach for the generation of infectiousvirus. The severe cytopathic effects of vaccinia virus and the safetyprecautions required for use of infectious agents are undesireablefeatures of this system. Use of the pol I-pol II system eliminates therequirement for a virus infection and allows the use of LLC-MK2 cellsfor transfection and growth of human Parainfluenza virus III, thusproviding a technology for generating attenuated viruses in a simplerand safer way.

FIG. 10. Plasmid-based system for the generation of Rotavirus fromcloned cDNA. This system can be used for generation of viruses withsegmented, double stranded RNA genomes (e.g., Rotavirus). It can beapplied, for example, to viruses which are members of the familyReoviridae (10, 11, 12 dsRNA segments) or Birnaviridae (2 dsRNAsegments). To date, for viruses of the family Reoviridae, no reversegenetics systems are available. This figure illustrates how rotaviruses,which have 11 dsRNA segments, may be generated using the presentinvention, but similar systems can be employed for members of the generaOrbivirus (10 dsRNA segments) or Orthoreoviruses (12 dsRNA segments).

The following discussion illustrates the generation of the rotavirusA/SA11 entirely from cloned cDNA. All 11 segments of the simianrotavirus double stranded RNA genome have been determined. The dsRNAs inthe genome are from 3302 bp to 663 bp long, and the size of the completegenome is 18,550 bp. The genome segments are numbered 1-11 in order ofincreasing mobility by PAGE (poly acrylamid gel electrophoresis)analysis. The segments are completely base-paired and the plus-sensestrand contains a 5′-terminal cap structure (m7GpppGmGPy) but does nothave a polyadenylation signal near its 3′-end. All genomic segmentsshare short conserved 5′ and 3′ termini with a 10 nucleotide consensusat the 5′-end and an 8 nucleotide consensus at the 3′-end. Immediatelyinternal to these terminal regions, in each gene, there is a secondregion of conservation of at least 30-40 nucleotides which aresegment-specific. The 5′-non translated regions (NTRs) vary in lengthbut are all less than 50 nucleotides and in all segments the NTRs arefollowed by at least one long open reading frame after the first AUG.Segments 9 and 11 encode two proteins. The 3′-NTRs vary in lengthranging from 17 nts (segment 1) to 182 nts (segment 10).

Rotavirus cDNA is cloned into a dual promoter system, preferably a polI-pol II system. After transfection of the resultant plasmids into asuitable host cell, viral RNAs and proteins are produced which resultsin formation of infectious rotavirus. Preferably, a unidirectionaltranscription system is used for producing rotavirus. Using thisapproach results in the intracellular synthesis of the 11 (+) RNAmolecules of the rotaviral genome which have triphosphates at their 5′termini. Expression of virus-like mRNA results in expression of viralproteins. The viral protein VP3(cap), which has a guanylyltransferaseand methyltransferase activity, catalyzes the addition of 5′-capstructures to all 11 rotaviral (+) RNA (Chen D., et al., Virology 1999,265:120-130). Indeed, it has been previously demonstrated that purifiedVP4(cap), a rotaviral VP3(cap) analogue of bluetogue virus (BTV), canadd cap structures to a viruslike (+) RNA in vitro (Ramadevi N., et al.Proc Natl Acad Sci USA. 1998, 95(23):13537-42). It is anticipated thatin vivo transcription of cDNA and VP3(cap) protein expressionintracellularly, results in the generation of capped RNAs for all 11rotaviral genomic segments. Those mRNAs are translated into viralproteins or are packaged into precore-RI . After the formation ofVP6(T13)-RI particles, the positive sense mRNA is used as template forthe synthesis of (−)RNA. Ultimately, the addition of VP4 and VP7 duringthe morphogenesis results in infectious progeny virions.

Since the efficient initiation of replication and morphogenesis may bedependent on the optimal concentration of each of the viral proteins, itmay be advantageous to generate separate plasmids for RNA synthesis andprotein expression. However, because the level of protein expression canbe optimized by varying the quantity of plasmids in the host cell or byuse of different promoters for mRNA synthesis, use of two plasmids forone segment is not likely to be necessary for most of the genes. Sincethe (+) RNA is synthesized from a (−) RNA, the intracellular expressionof (−) RNA and protein may result in the generation of replicationcompetent units, which produce viral mRNA. Thus, the dual promotersystem allows the establishment of a minimum plasmid system comprising asignificantly lower number of plasmids than 22 which would be necessaryif the RNA and protein expressing plasmids are on separate plasmids.Rotaviral generation may be performed in a similar manner to that usedto produce influenza A virus.

FIGS. 11A-D. Replication and mRNA synthesis of RNA virus genomes.

-   (A) mRNAs are synthesized by the viral polymerase proteins during    infection of (−) strand viruses: One or two mRNAs for segmented RNA    viruses or multiple mRNAs for viruses with unimolecular genomes.    Antitermination mechanisms result in the synthesis of full length    (+) strands, which can be copied into (−) genomic RNA.-   (B) The segmented genomes of ambisense RNA viruses are copied to    form one mRNA; a second RNA is synthesized from the complement.-   (C) In cells infected with double-stranded RNA viruses, the mRNAs    first synthesized can either be translated into protein or serve as    templates for the synthesis of (−) strands, resulting in    double-stranded genomic RNA.-   (D) For (+) strand viruses, the genomic RNA is also an mRNA and is    copied into (−) strand RNA, which can be copied into (+) genomic    RNA. The mRNAs of some (+) RNA viruses do not contain a polyA tail.    In some families one or more subgenomic RNAs are produced.

DETAILED DESCRIPTION

The life cycle of all RNA viruses includes RNA synthesis and assembly ofvirus particles after protein synthesis; these functions provide aconceptual framework for the reverse genetics systems of the presentinvention which may be used to produce RNA viruses from cloned cDNA. Thepresent invention simplifies and improves currently available reversegenetics systems by establishing a dual promoter system for theproduction of negative strand segmented viruses (e.g., influenza A,influenza B, Bunyaviridae), nonsegmented negative strand RNA viruses(e.g., Paramyxoviridae, Mononegavirales), double strand RNA viruses(e.g., Reoviridae, Birnaviridae) and positive strand RNA viruses (e.g.,Flaviviridae, Picornaviridae, Coronaviridae, Togaviridae). Because thesystem of the present invention uses a single viral cDNA for bothprotein synthesis and genomic RNA synthesis, this systems reduces thenumber of plasmids required for virus production and allows thedevelopment of vaccines quickly and cheaply.

If a virus comprising a segmented RNA genome is to be produced using thepresent invention, a viral cDNA corresponding to each gene in the targetgenome is inserted into an expression plasmid of the invention. Theinvention comprises a bidirectional plasmid based expression system anda unidirectional plasmid based expression system wherein viral cDNA isinserted between an RNA polymerase I (pol I) promoter and terminatorsequences (inner transcription unit). This entire pol I transcriptionunit is flanked by an RNA polymerase II (pol II) promoter and apolyadenylation site (outer transcription unit). In the unidirectionalsystem, the pol I and pol II promoters are upstream of the cDNA andproduce positive-sense uncapped cRNA (from the pol I promoter) andpositive-sense capped mRNA (from the pol II promoter). The pollpromoter, pol I terminator sequence, pol II promoter and polyadenylationsignal in the unidirectional system may be referred to as comprising an“upstream-to-downstream orientation”. In the bidirectional system, thepol I and pol II promoters are on opposite sides of the cDNA wherein anupstream pol II promoter produces positive-sense capped mRNA and adownstream pol I promoter produces negative-sense uncapped viral RNA(vRNA). These pol I-pol II systems start with the initiation oftranscription of the two cellular RNA polymerase enzymes from their ownpromoters, presumably in different compartments of the nucleus. The polI promoter and pol I terminator sequence in the bidirectional system maybe referred to as comprising a “downstream-to-upstream orientation”whereas the pol II promoter and polyadenylation signal in thebidirectional system may be referred to as comprising an“upstream-to-downstream orientation”.

If the target virus comprises a positive strand, segmented RNA genome, apol I promoter is, preferably, located upstream of the cDNA in the innertranscription unit (unidirectional system). In this embodiment, positivestrand RNA is generated for direct incorporation into new viruses.However, embodiments wherein target viruses comprise negative strand,segmented RNA genomes are produced using the unidirectional system arewithin the scope of the invention.

If the target virus comprises a negative strand, segmented RNA genome,the pol I promoter is, preferably, located downstream of the cDNA in theinner transcription unit (bidirectional system). In this embodiment,negative stranded RNA is generated for direct incorporation into newviruses. Embodiments wherein target viruses comprising positivestranded, segmented RNA genomes are produced with the bidirectionalsystem are within the scope of the invention.

The present invention may also be used to produce viruses comprisinginfectious or uninfectious unsegmented RNA genomes (single stranded ordouble stranded). In general, simple introduction of infectious viralgenomic RNA into a host cell is sufficient to cause initiation of theviral life cycle within the cell and the eventual production of completeviruses. For example, simple introduction of picornaviral genomic RNAinto a host cell is sufficient to cause generation of completepicomaviruses. Initiation of the life cycle of a virus comprisinguninfectious genomic RNA, typically, requires the additionalintroduction of other viral proteins which are usually carried withinthe viral particle along with the genome. For example, parainfluenzavirus III carries an RNA dependent RNA polymerase whose presence isrequired within a newly infected host cell for initiation of viralgenomic RNA replication and transcription of viral mRNAs; in the absenceof the polymerase, parainfluenza III genomic RNA is not infectious. Inembodiments of the present invention wherein viruses comprisinginfectious, unsegmented genomic RNAs are generated, simple introductionof a dual expression plasmid of the invention, carrying a nucleic acidincluding the viral genome, into a suitable host cell is sufficient tocause generation of complete viruses. In embodiments wherein virusescomprising uninfectious unsegmented genomic RNA are generated,additional expression plasmids may also have to be introduced into ahost cell along with the dual expression plasmid carrying the viralgenome. The additional plasmid should express the protein(s) requiredfor initiation of the viral life cycle which are normally introducedinto a host cell upon infection (e.g., RNA dependent RNA polymerases).

In embodiments wherein picornavirus, which comprising an infectious,unsegmented RNA genome, is produced, cDNA comprising the complete viralgenome is inserted into a dual promoter expression plasmid of theinvention. An upstream promoter in an outer transcription unit,preferably, a pol II promoter, directs production of a positive strandmRNA comprising the complete viral genome—a polyprotein is translatedfrom the mRNA and individual proteins are cleaved and liberated from thepolyprotein (e.g., by a protease within the polyprotein). Since theviral genome comprises positive strand RNA, a second upstream promoterin an inner transcription unit (unidirectional system), preferably polI, directs production of a positive stranded copy of the genome. If theviral genome comprised negative strand RNA, a second downstreampromoter, in an inner transcription unit (bidirectional system),preferably pol I, would direct production of a negative stranded copy ofthe genome. Embodiments wherein negative stranded, unsegmented RNAviruses are produced using the unidirectional system are within thescope of the invention. Similarly, embodiments wherein positivestranded, unsegmented RNA viruses are produced using the bidirectionalsystem are within the scope of the invention.

Viruses comprising uninfectious, unsegmented RNA genomes wherein apolyprotein is not produced can also be generated with the presentinvention. For example, the present system may be used to producerhabdoviridae viruses or paramyxoviridae viruses, preferablyparainfluenza virus III, whose life cycle normally includes productionof multiple monocistronic mRNAs from genomic, negative strand RNA by avirally derived RNA dependent RNA polymerase; individual proteins areexpressed from the monocistronic mRNAs. In these embodiments, an outertranscription unit comprising a promoter, preferably a pol II promoter,directs production of a positive strand, polycistronic copy of the viralgenome from which, generally, only the first gene (NP) is translated.Additionally, an inner transcription unit comprising a promoter,preferably a pol I promoter, directs expression of an RNA copy of thegenome for incorporation into new viruses. Since the parainfluenza IIIviral genome comprises negative stranded RNA, the promoter of the innertranscription unit is preferably located downstream of the cDNA(bidirectional system). If the viral genome comprises positive strandRNA, the promoter of the inner transcription unit is preferably locatedupstream of the cDNA (unidirectional system). Embodiments whereinviruses comprising a positive stranded RNA genome are produced using thebidirectional system and embodiments wherein viruses comprising anegative stranded RNA genome are produced using the unidirectionalsystem are within the scope of the invention. Additional viral proteins(other than the protein expressed from the polycistronic mRNA) arerequired for viral transcription and replication (L and P), and theseproteins are provided individually on separate expression plasmids.

The invention may also include embodiments wherein viruses comprisingdouble stranded, segmented RNA genomes are generated. In theseembodiments, a plasmid comprising each gene in the target viral genomeis inserted into a dual promoter expression plasmid of the invention.The plasmid may be either a unidirectional plasmid or a bidirectionalplasmid. A promoter in an outer transcriptional unit, preferably a polII promoter, directs expression of an mRNA transcript of each gene whichis translated into the encoded protein. A promoter in an innertranscription unit, preferably a pol I promoter, directs transcriptionof either a positive strand (unidirectional system) or a negative strand(bidirectional system). Subsequently, the first strand which is producedmay act as a template for production of the complementary strand byviral RNA polymerase. The resulting double stranded RNA product isincorporated into new viruses.

Recovery of the two influenza A viruses from a minimal plasmid-basedsystem, A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1), establishedutility of this system. Recovery of a phenotypically indistinguishableA/PR/8/34 (H1N1) strain, which is the standard for production ofinactivated influenza A vaccine, established the usefulness of thissystem for vaccine development. Seventy-two hours after the transfectionof eight expression plasmids into co-cultured 293T and MDCK cells, thevirus yield in the supernatant of the transfected cells was between2×10⁵ and 2×10⁷ infectious viruses per ml. This eight-plasmid system wasalso used to generate single and quadruple reassortment viruses betweenA/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1), and to generate A/WSN/33viruses from a tandem oriented system (which produces cRNA and mRNA).

Because the pol I-pol II system facilitates the design and recovery ofboth recombinant and reassortment influenza A viruses, it is alsoapplicable to the recovery of other RNA viruses entirely from clonedcDNA. Although cDNA is preferred for use in the present invention, anyother type of nucleic acid which encodes a viral gene which is to beexpressed may be used if the essential elements of the invention arepreserved. For example, PCR amplified products or restriction fragmentscomprising viral genes may be used. Furthermore, the genes expressed inthe plasmid based system of the invention may be fused to or tagged withother genes such as purification/detection tags (e.g., glutathione-Stransferase, polyhistidine, green fluorescent protein, myc tags and FLAGtags). The present invention also anticipates embodiments whereinpartial gene sequences are used in plasmids of the present system.

The following table includes a non-limiting list of negative strandedRNA viruses which may be produced using the present invention: OrderFamily Subfamily Genus Type Species Mononegavirales BornaviridaeBornavirus Borna disease virus Mononegavirales Filoviridae Ebola-likevirus Ebola virus Mononegavirales Filoviridae Marburg-like Marburg virusvirus Mononegavirales Paramyxoviridae Paramyxovirinae Respirovirus Humanparainfluenza virus 1 Mononegavirales Paramyxoviridae ParamyxovirinaeMorbillivirus Measles virus Mononegavirales ParamyxoviridaeParamyxovirinae Rubulavirus Mumps virus Mononegavirales ParamyxoviridaePneumovirinae Pneumovirus Human Respiratory Syncitial VirusMononegavirales Paramyxoviridae Pneumovirinae Metapneumovirus TurkeyRhinotracheitis Virus Mononegavirales Rhabdoviridae VesiculovirusVesicular Stomatitis Indiana Virus Mononegavirales RhabdoviridaeLyssavirus Rabies Virus Mononegavirales Rhabdoviridae EphemerovirusBovine Ephemeral Fever Virus Mononegavirales RhabdoviridaeNovirhabdovirus Infectious Hematopoietic Necrosis Virus MononegaviralesRhabdoviridae Cytorhabdovirus Lettuce Necrotic Yellows VirusMononegavirales Rhabdoviridae Nucleorhabdovirus Potato Yellow DwarfVirus Mononegavirales Orthomyxoviridae Influenzavirus A Influenza AVirus Mononegavirales Orthomyxoviridae Influenzavirus B Influenza BVirus Mononegavirales Orthomyxoviridae lnfluenzavirus C Influenza CVirus Mononegavirales Orthomyxoviridae Thogotovirus Thogoto VirusMononegavirales Bunyaviridae Bunyavirus Bunyamwera Virus MononegaviralesBunyaviridae Hantavirus Hantaan Virus Mononegavirales BunyaviridaeNairovirus Nairobi Sheep Disease Virus Mononegavirales BunyaviridaePhlebovirus Sandfly Fever Sicilian Virus Mononegavirales BunyaviridaeTospovirus Tomato Spotted Wilt Virus Mononegavirales BunyaviridaeTenuivirus Rice Stripe Virus Mononegavirales Bunyaviridae OphiovirusCitrus Psorosis Virus Mononegavirales Arenaviridae ArenavirusLymphocytic Chorio-meningitis Virus Mononegavirales ArenaviridaeDeltavirus Hepatitis Delta Virus

The present invention is further based, in part, on development of abidirectional transcription construct that contains viral cDNA encodingPB2, PB1, PA, HA, NP, NA, M or NS flanked by an RNA polymerase I (pol I)promoter for vRNA synthesis and an RNA polymerase II (pol II) promoterfor viral mRNA synthesis. The utility of this approach is proved by thegeneration of virus after transfecting the pol I/pol II-promoter-PB1construct together with vRNA- and protein-expression constructs for theremaining seven segments. Because this approach reduces the number ofplasmids required for virus generation, it also reduces the worknecessary for cloning, enhances the efficiency of virus generation andexpands the use of the reverse genetics system to cell lines for whichefficient cotransfection of 17 plasmids can not be achieved. Alsoincluded is a unidirectional transcriptional construct comprising pol Iand pol II promoters placed upstream of a viral cDNA coding sequence.Both promoters are in a common upstream-to-downstream orientation inrelation to the gene. Although the bidirectional system produces ahigher influenza A viral titer, the unidirectional system is useful forother applications (e.g., production of other negative strand viralstrains).

A promoter, terminator or polyadenylation signal is “upstream” of a geneif it is proximal to the start of the gene (e.g., the first codon) anddistal to the end of the gene (e.g., the termination codon). A promoter,terminator or polyadenylation signal is “downstream” of a gene if it isproximal to the end of the gene and distal to the start of the gene.Promoters in the plasmids of the invention, which are functionallyassociated with a gene, are oriented so as to promote transcription of asense or an antisense strand of the gene.

As used herein “expression plasmid” is a DNA vector comprising an “innertranscription unit” and an “outer transcription unit”. As discussedabove, expression plasmids may be used to generate any type of RNAvirus, preferably positive or negative strand RNA viruses, segmented orunsegmented genome RNA viruses or double stranded RNA viruses. The outertranscription unit comprises a promoter, preferably a pol II promoter,which directs transcription, from a viral cDNA, of mRNA which istranslatable. The outer transcription unit may comprise a T7 RNApolymerase promoter, a T3 RNA polymerase promoter, an SP6 RNA polymerasepromoter or any promoter or combination of genetic elements capable ofdriving expression of a translatable RNA product. For example, the outertranscription unit may comprise a pol I or pol II promoter if the viralcDNA, which is to be expressed from the plasmid, is geneticallyengineered to include a polyadenylation signal at the 3′ end of thetranscribed RNA. This may be accomplished by inserting a string of Anucleotides at the 3′ end of the cDNA coding sequence immediately beforethe stop codon. Furthermore, the viral cDNA can be geneticallyengineered to include an Internal Ribosomal Entry Site (IRES) at the 5′end of the cDNA to facilitate translational initiation from thetranscribed RNA. An “inner transcription unit” in the expressionplasmids of the invention is located within the outer transcription unitand comprises a promoter, preferably a pol I promoter, which can directtranscription of a viral cDNA which may be replicated by viral machineryand incorporated into new viruses. Preferably the promoter in the innertranscription unit transcribes RNA which does not comprise excess,non-virus extraneous, sequences at the 5′ and 3′ ends, preferably byprecise fusion of viral cDNA with pol I promoter and terminatorsequences. However, the invention includes embodiments wherein the innertranscription unit comprises a promoter which does direct production ofRNA transcripts comprising 5′ and 3′ additional non-virus sequence(e.g., pol II promoters, pol III promoters, T7 RNA polymerase promoter,T3 RNA polymerase promoters or SP6 RNA polymerase promoters). In theseembodiments, additional sequences may be included in the expressionplasmid which ensure that RNA which is produced from the innertranscription unit do not include excess non-virus sequence. Forexample, the expression plasmid may be genetically engineered to includeribozymal sequences at the ends of transcripts produced from the innertranscription unit wherein the ribozymal portions of the transcribed RNAcleave the transcript in such a way that the sequences of the 5′ and 3′termini of the RNAs are generated as found in the virus RNA.Furthermore, the expression plasmids may be genetically engineered toinclude terminator sequences which cause transcriptional termination atthe end of the viral cDNA coding sequence thereby preventingincorporation of excess untranslatable sequences at the 3′ end of thetranscribed RNA. Preferably, an “expression plasmid” is a DNA vectoremploying the pol I-pol II system. Thus, such a plasmid comprises an RNApolymerase I (pol I) promoter and pol I terminator sequences, which areinserted between an RNA polymerase II (pol II) promoter and apolyadenylation signal. In the bidirectional system, an RNA polymerase Ipromoter controls expression of genomic negative sense uncapped RNA(termed herein “vRNA”) of the cDNA which is inserted in an “antisense”orientation between the promoter and terminator. An RNA polymerase IIpromoter controls expression of messenger RNA; a viral gene cDNA segmentinserted in a “sense” orientation between the pol II promoter andpolyadenylation signal results in expression of positive-sense cappedviral mRNA. In the unidirectional system, the viral cDNA is inserteddownstream of the pol I and pol II promoters in a sense orientation. Thepol II promoter drives the expression of positive-sense capped viralmRNA and the pol I promoter drives the expression of positive senseuncapped viral cRNA.

A plasmid may comprise “essentially all” of another base plasmid if allof the genes and functional non-coding regions of the base plasmid arepresent. For example, a plasmid constructed by mere removal of a portionof a polylinker and insertion of a foreign gene comprises essentiallyall of the base plasmid.

A “negative strand RNA virus” is a virus in which the viral genomecomprises negative strand RNA. Negative strand RNA is complementary tomRNA and, generally, must be copied into the complementary positivestrand mRNA for proteins to be translated. Typically, these virusespackage an RNA dependent RNA polymerase for production of mRNA upon hostcell infection. Negative strand RNA virus families include, but are notlimited to, Orthomyxoviridae, Arenaviridae, and Bunyaviridae.Preferably, the viral genome is from a virus that is a member of theOrthomyxoviridae virus family, and optimally has a segmented genome.Members of the Orthomyxovirdae virus family include but are not limitedto influenza A, influenza B, influenza C, Thogotovirus, Measles andMumps viruses (Paramyxovirus) or Rhabies virus (Rhabdovirus).

A “positive strand RNA virus” is a virus comprising a positive strandRNA genome. Examples of positive stranded RNA viruses include Poliovirus(Picornavirus), Togaviruses and Flaviviruses. The genomic RNA of theseviruses is the same sense as mRNA and may function as mRNA. Theseviruses may comprise a segmented or unsegmented genome.

A “double stranded RNA virus” comprises a double stranded RNA genome.Reoviruses are double stranded RNA viruses.

A viral genome may also be segmented or unsegmented (unimolecular). Asegmented genome comprises two or more nucleic acids each encoding oneor more viral genes. Preferably, a segmented genome comprises a separatenucleic acid for each viral gene. Orthomyxoviruses (Influenza A, B or Cvirus), Bunyaviruses and Arenaviruses comprise segmented RNA genomes. Anonsegmented genome comprises a single nucleic acid comprising everyviral gene. Mononegavirales viruses, Rhabdoviruses, Flaviviridaeviruses, Picornaviridae viruses, Coronaviridea viruses, Togaviridaeviruses and Paramyxoviruses comprise a nonsegmented RNA genome.

Double stranded RNA may be abbreviated as “dsRNA”. Single stranded RNAmay be abbreviated as “ssRNA.” Single, negative strand RNA may beabbreviated as “−ssRNA”. Single, positive strand RNA may be abbreviatedas “+ssRNA.”

A “viral gene segment” is, preferably, a cloned cDNA corresponding to agenomic RNA molecule from an RNA virus genome. This term may alsoinclude any gene or gene segment (e.g., a PCR product or restrictionfragment) comprising a gene derived from an RNA virus.

A “minimum plasmid-based system” is a system in which there is anexpression plasmid, as defined above, containing each autonomous viralgenomic segment from an RNA virus. Thus, the total number of plasmidswhich contain viral genomic sequences will not exceed the total numberof gene segments from the source RNA virus. The invention includesembodiments wherein other plasmids, which do not contain viralsequences, may optionally be cotransfected into the host cells. Thisprovides significant advantages, by limiting the total number ofplasmids required to establish the system in a host cell, eliminatingthe need for helper virus, eliminating the need for a selection process,and permitting efficient generation of reassortment viruses.

Certain viral genes in a minimum plasmid-based system of the inventioncan be from a viral strain well adapted to grow in cell culture, such asthe PR/8/34 (H1N1) or WSN/33 (H1N1) strain, or from an attenuatedstrain, such as the A/Ann Arbor/6/60 (H2N2), or for influenza B/AnnArbor/1/66. Preferably, an attenuated strain is also well adapted forgrowth in cell culture. In particular, for influenza A, preferred viralgene segments in the plasmid-based system encode viral polymerasecomplex proteins, M proteins, and/or NS proteins from a strain welladapted to grow in cell culture or from an attenuated strain, or both.These proteins are termed herein “viral internal proteins” or viral“non-glycoprotein”.

The genome of influenza A virus typically encodes 10 different proteins:PB2, which is believed to be a transcriptase, PB1, which is believed tobe a transcriptase, PA which is believed to be a transcriptase, HA whichis believed to be hemeagglutinin, NP, which is believed to be an RNAbinding nucleoprotein, NA which is believed to be neuraminidase, M1/M2which are believed to be a matrix protein and an integral membraneprotein, respectively, and NS1/NS2 which are believed to benonstructural proteins which may affect RNA processing and transport.PB1, PB2, NP and PA are believed to be part of an influenza virustranscriptional polymerase complex.

The term “cell culture” as used herein preferably refers to acommercially acceptable method for propagating virus for vaccineproduction, e.g., embryonated hens' eggs, as well as to cell culture invitro in a host cell (see Furminger, In: Nicholson, Webster and May(eds.), Textbook of Influenza, Chapter 24, pp. 324-332, particularly pp.328-329).

Similarly, the plasmid-based system will incorporate gene segments forantigens required to produce a protective immunological response. A“protective immunological response” comprises a humoral (antibody) orcellular component, or both, effective to eliminate virions and infectedcells in an immunized (vaccinated) subject. Thus, a protective immuneresponse can prevent or resolve an RNA virus, e.g., influenza virus,infection. Preferably, the antigens are “surface antigens”, i.e.,expressed on the surface of the virion or the surface of infected cells.More preferably, the surface antigens are glycoproteins. For influenza,the primary glycoprotein antigens are hemagluttinin (HA or H) andneuraminidase (NA or N).

As used herein, the term “immunogenic” means that the polypeptide iscapable of eliciting a humoral or cellular immune response, andpreferably both. An immunogenic entity is also antigenic. An immunogeniccomposition is a composition that elicits a humoral or cellular immuneresponse, or both, when administered to an animal. A molecule is“antigenic” when it is capable of specifically interacting with anantigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains an epitope of at least about five, and preferablyat least about 10, amino acids. An antigenic portion of a polypeptide,also called herein the epitope, can be that portion that isimmunodominant for antibody or T cell receptor recognition, or it can bea portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier polypeptide for immunization. Amolecule that is antigenic need not be itself immunogenic, i.e., capableof eliciting an immune response without a carrier.

The term “pathogenic virus strain” is used herein to refer to any virusstrain that is capable of causing disease; preferably, the virus is onthe current World Health Organization (WHO), Centers for Disease Controland Prevention (CDC), or other public health authority list of likelycirculating viruses. Such viruses may include members of the hepatitisvirus family, reovirus family, orthomyxovirus family, the paramyxovirusfamily, the filoviridae family, the bornaviridae family, thebunyaviridae family, the arenaviridae family, coronaviridae family,poliovirus family or the rhabdovirus family. The inventionadvantageously provides for inserting the genes for the primary antigensfrom such strains into a plasmid-based system, in which the remainingviral genes have desired culture and/or attenuation characteristics,thus providing for production of quantities of virus of the appropriateantigenic background for vaccine production. For example, the genes ofthe paramyxovirus, human parainfluenza virus 3 (nucleocapsid gene,phosphoprotein gene, matrix gene, fusion gene,hemagglutinin-neuraminidase protein gene and large gene), mayconveniently be placed in the plasmids of the invention for productionof parainfluenza virus 3 virions.

Thus, a preferred “reassortment” virus of the invention is a virus inwhich gene segments encoding antigenic proteins from a pathogenic virusstrain are combined with gene segments encoding viral polymerase complexor other similar genes (e.g., non-glycoprotein genes, including M genesand NS genes) from viruses adapted for growth in culture (or attenuatedviruses). The reassortment virus thus carries the desired antigeniccharacteristics in a background that permits efficient production in ahost cell, as described above. Such a reassortment virus is a desirable“virus seed” for production of virions to produce vaccine (seeFurminger, supra).

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of recombinant RNA virions, preferably negative strandsegmented RNA virions, by the cell. Exemplary host cells include, butare not limited to, Madin-Darby Canine Kidney (MDCK) cells, VERO cells,CV1 cells, COS-1 and COS-7 cells, and BHK-1 cells, for example and notby way of limitation. In a specific embodiment, a transient co-cultureis preferred for producing virions. Co-culturing permits efficienttransfection of a receptive cell, such as a 293T cell, with subsequentinfection of a permissive cell for viral growth, such as an MDCK cell.

The term “RNA virus virions” refers to the viral particles, which whenfirst produced are fully infectious, from host cells transfected orco-transfected with a plasmid-based system of the invention. Such asystem produces vRNA and viral proteins (from viral mRNA translation),resulting in assembly of infectious viral particles (virions).

As used herein, a “RNA virus-specific vaccine” is a composition that canelicit protective immunity to an RNA virus when administered to asubject. The term “vaccine” refers to a composition containing virus,inactivated virus, attenuated virus, split virus, or viral protein,i.e., a surface antigen, that can be used to elicit protective immunityin a recipient (see Furminger, supra). It should be noted that to beeffective, a vaccine of the invention can elicit immunity in a portionof the population, as some individuals may fail to mount a robust orprotective immune response, or, in some cases, any immune response. Thisinability may stem from the individual's genetic background or becauseof an immunodeficiency condition (either acquired or congenital) orimmunosuppression (e.g., treatment with immunosuppressive drugs toprevent organ rejection or suppress an autoimmune condition). Efficacycan be established in animal models.

A “protective dose” of a vaccine is an amount, alone or in conjunctionwith an adjuvant, effective to elicit a protective immune response in arecipient subject. Protection can also depend on the route ofadministration, e.g., intramuscular (preferred for an inactivatedvaccine) or intranasal (preferred for an attenuated vaccine).

The term “subject” as used herein refers to an animal that supports anRNA virus infection, including, but not limited to, water fowl,chickens, pigs, and humans. In particular, the term refers to a human.

An “adjuvant” is a molecule or composition that potentiates the immuneresponse to an immunogen. An adjuvant is “acceptable for use in a human”when it is pharmaceutically acceptable, as defined below. Examples ofadjuvants are provided below.

As used herein, the term “isolated” means that the referenced materialis removed from its native environment, e.g., a cell. Thus, an isolatedbiological material can be free of some or all cellular components,i.e., components of the cells in which the native material occursnaturally (e.g., cytoplasmic or membrane component). A material shall bedeemed isolated if it is present in a cell extract or supernatant. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an isolated mRNA, a cDNA, or a restriction fragment. Inanother embodiment, an isolated nucleic acid is preferably excised fromthe chromosome in which it may be found, and more preferably is nolonger joined or proximal to non-coding regions (but may be joined toits native regulatory regions or portions thereof), or to other genes,located upstream or downstream of the gene contained by the isolatednucleic acid molecule when found in the chromosome. In yet anotherembodiment, the isolated nucleic acid lacks one or more introns.Isolated nucleic acid molecules include sequences inserted intoplasmids, cosmids, artificial chromosomes, and the like, i.e., when itforms part of a chimeric recombinant nucleic acid construct. Thus, in aspecific embodiment, a recombinant nucleic acid is an isolated nucleicacid. An isolated protein may be associated with other proteins ornucleic acids, or both, with which it associates in the cell, or withcellular membranes if it is a membrane-associated protein. An isolatedorganelle, cell, or tissue is removed from the anatomical site in whichit is found in an organism. An isolated material may be, but need notbe, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified virion ispreferably substantially free of host cell or culture components,including tissue culture or egg proteins, non-specific pathogens, andthe like. As used herein, the term “substantially free” is usedoperationally, in the context of analytical testing of the material.Preferably, purified material substantially free of contaminants is atleast 50% pure; more preferably, at least 90% pure, and more preferablystill at least 99% pure. Purity can be evaluated by chromatography, gelelectrophoresis, immunoassay, composition analysis, biological assay,and other methods known in the art.

Methods for purification are well-known in the art. Viral particles canbe purified by ultrafiltration or ultracentrifugation, preferablycontinuous centrifugation (see Furminger, supra). Other purificationmethods are possible and contemplated herein. A purified material maycontain less than about 50%, preferably less than about 75%, and mostpreferably less than about 90%, of the cellular components, media,proteins, or other nondesirable components or impurities (as contextrequires), with which it was originally associated. The term“substantially pure” indicates the highest degree of purity which can beachieved using conventional purification techniques known in the art.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, and more preferably within 5% of agiven value or range. Alternatively, logarithmic terms used in biology,the term “about” can mean within an order of magnitude of a given value,and preferably within one-half an order of magnitude of the value.

Genetic Engineering of Plasmid-Based Systems

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

These routine techniques apply to the preparation of pol I-pol IIplasmid systems, isolation of viral gene segment cDNA clones, insertionof such DNAs into plasmids, and transfection of cells with a plasmid orplasmid-based system of the invention. In particularly, routinetechniques of site-directed mutagenesis or gene modification permitmodification of the RNA viral, preferably, negative strand segmented RNAviral genes to develop attenuated virus, as set forth below; or viralproteins that incorporate novel epitopes, e.g., in the neuraminidasestalk; or to create defective viruses.

“Amplification” of DNA as used herein denotes the use of polymerasechain reaction (PCR) to increase the amount of a particular DNA sequencewithin a mixture of DNA sequences. For a description of PCR see Saiki etal., Science 1988, 239:487.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. A “recombinant DNAmolecule” is a DNA molecule that has undergone a molecular biologicalmanipulation.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in DNA and RNA, and means any chain oftwo or more nucleotides. A nucleotide sequence typically carries geneticinformation, including the information used by cellular machinery tomake proteins.

The polynucleotides herein may be flanked by heterologous sequences,including promoters, internal ribosome entry sites (IRES; Ghattas, etal., Mol. Cell. Biol. 11:5848-5859, 1991) and other ribosome bindingsite sequences, enhancers, response elements, suppressors, signalsequences, polyadenylation sequences, introns, 5′- and 3′-non-codingregions, and the like. The nucleic acids may also be modified by manymeans known in the art. Non-limiting examples of such modificationsinclude methylation, “caps” such as 5′-7-methyl-G(5′)ppp(5′)N caps,substitution of one or more of the naturally occurring nucleotides withan analog, and internucleotide modifications. Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

A “coding sequence” or a sequence “encoding” an expression product, suchas a polypeptide, is a nucleotide sequence that, when expressed, resultsin the production of that polypeptide, i e., the nucleotide sequenceencodes an amino acid sequence for that polypeptide. A coding sequencefor a protein may include a start codon (usually ATG) and a stop codon.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more polypeptides, and may or maynot include regulatory DNA sequences, such as promoter sequences, whichdetermine for example the conditions under which the gene is expressed.

In addition, the present invention permits use of various mutants,sequence conservative variants, and functionally conservative variantsof RNA virus gene segments, preferably negative strand RNA virus genesegments, provided that all such variants retain the requiredimmunoprotective effect. Indeed, the invention advantageously permitsmutagenesis to develop attenuated viral strains in a systematic fashion.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, or any process, mechanism, or result of such achange. This includes gene mutations, in which the structure (e.g. DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g. protein) expressed bya modified gene or DNA sequence. The term “variant” may also be used toindicate a modified or altered gene, DNA sequence, enzyme, cell, etc.,i.e., any kind of mutant. Mutations can be introduced by randommutagenesis techniques, or by site-directed mutagenesis, includingPCR-based sequence modification. As noted above, and discussed in detailbelow, mutagenesis of one or more individual gene segments of an RNAvirus (e.g., a negative strand segmented RNA virus) permits developmentof attenuated viruses, as well as elucidation of the attenuationmechanism. Moreover, the plasmid-based system of the invention overcomesthe drawbacks of prior efforts to develop attenuated viruses bymutagenesis, such as the restrictions of an efficient selection system(see Bilsel and Kawaoka, In: Nicholson, Webster and May (eds.), Textbookof influenza, Chapter 32, pp. 422-434, especially pp. 423-425).

“Sequence-conservative variants” of a polynucleotide sequence are thosein which a change of one or more nucleotides in a given codon positionresults in no alteration in the amino acid encoded at that position.Allelic variants can be sequence-conservative variants.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Some allelicvariations result in functional-conservative variants, such that anamino acid substitution does not dramatically affect protein function.Similarly, homologous proteins can be function-conservative variants.Amino acids with similar properties are well known in the art. Forexample, arginine, histidine and lysine are hydrophilic-basic aminoacids and may be interchangeable. Similarly, isoleucine, a hydrophobicamino acid, may be replaced with leucine, methionine or valine. Suchchanges are expected to have little or no effect on the apparentmolecular weight or isoelectric point of the protein or polypeptide.Amino acids other than those indicated as conserved may differ in aprotein or enzyme so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and maybe, for example, from 70% to 99% as determined according to an alignmentscheme such as by the Cluster Method, wherein similarity is based on theMEGALIGN algorithm. A “function-conservative variant” also includes apolypeptide or enzyme which has at least 60% amino acid identity asdetermined by BLAST or FASTA algorithms where the parameters areselected to give the largest match between the sequences tested, overthe entire length of the reference sequence, preferably at least 75%,most preferably at least 85%, and even more preferably at least 90%, andwhich has the same or substantially similar properties or functions asthe native or parent protein or enzyme to which it is compared.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reeck,et al., Cell 50:667, 1987). Such proteins (and their encoding genes)have sequence homology, as reflected by their sequence similarity,whether in terms of percent similarity or the presence of specificresidues or motifs.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck, et al., supra). However, in common usageand in the instant application, the term “homologous,” when modifiedwith an adverb such as “highly,” may refer to sequence similarity andmay or may not relate to a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when a sufficient number of thenucleotides match over the defined length of the DNA sequences todifferentiate the sequences from other sequences, as determined bysequence comparison algorithms, such as BLAST, FASTA, DNA Strider, andothers where parameters are selected to give the largest match betweenthe sequences tested, over the entire length of the reference sequence.Sequences that are substantially homologous can be identified bycomparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Such stringent conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989), 6.3.1-6.3.6. A non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., and more preferablyat 60° C. or 65° C.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when enough of theamino acids are identical or similar (functionally identical) over adefined length to differentiate the sequences from other sequences.Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program, orany of the programs described above (BLAST, FASTA, etc.). The followingreferences regarding the BLAST algorithm are herein incorporated byreference: BLAST ALGORITHMS: Altschul, S. F., Gish, W., Miller, W.,Myers, E. W. & Lipman, D. J., J. Mol. Biol. 215:403-410, 1990; Gish, W.& States, D. J., Nature Genet. 3:266-272, 1993; Madden, T. L., Tatusov,R. L. & Zhang, J., Meth. Enzymol. 266:131-141, 1996; Altschul, S. F.,Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. &Lipman, D. J., Nucleic Acids Res. 25:3389-3402, 1997; Zhang, J. &Madden, T. L., Genome Res. 7:649-656, 1997; Wootton, J. C. & Federhen,S., Comput. Chem. 17:149-163, 1993; Hancock, J. M. & Armstrong, J. S.,Comput. Appl. Biosci. 10:67-70, 1994; ALIGNMENT SCORING SYSTEMS:Dayhoff, M. O., Schwartz, R. M. & Orcutt, B. C. (1978) “A model ofevolutionary change in proteins.” In “Atlas of Protein Sequence andStructure”, vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl.Biomed. Res. Found., Washington, D.C.; Schwartz, R. M. & Dayhoff, M. O.(1978) “Matrices for detecting distant relationships.” In “Atlas ofProtein Sequence and Structure”, vol. 5, suppl. 3. M. O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S.F., J. Mol. Biol. 219:555-565, 1991; States, D. J., Gish, W., Altschul,S. F., Methods 3:66-70, 1991; Henikoff, S. & Henikoff, J. G., Proc.Natl. Acad. Sci. USA 89:10915-10919, 1992; Altschul, S. F., J. Mol.Evol. 36:290-300, 1993; ALIGNMENT STATISTICS: Karlin, S. & Altschul, S.F., Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990; Karlin, S. &Altschul, S. F., Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993; Dembo,A., Karlin, S. & Zeitouni, O., Ann. Prob. 22:2022-2039, 1994 andAltschul, S. F. (1997) “Evaluating the statistical significance ofmultiple distinct local alignments.” In “Theoretical and ComputationalMethods in Genome Research.” (S. Suhai, ed.), pp. 1-14, Plenum, NewYork.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a sequence. Forpurposes of defining the present invention, a promoter sequence which islocated upstream of a cDNA is bounded at its 3′ terminus by atranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. A promoter sequencewhich is located downstream of a cDNA (to express a (−)RNA) is boundedat its 5′ terminus by a transcription initiation site and extendsdownstream (3′ direction) to include the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. The bidirectional system of the invention includes bothupstream and downstream promoters; the unidirectional system includesonly upstream promoters. Within the promoter sequence will be found atranscription initiation site (conveniently defined for example, bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase.

Any known promoter may be used in the present invention as long as theessential elements of the invention are preserved. For example, pol IIpromoters that may be used to control gene expression include, but arenot limited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839and 5,168,062), the SV40 early promoter region (Benoist and Chambon,Nature 290:304-310, 1981), the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell22:787-797, 1980), the herpes thymidine kinase promoter (Wagner, et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42, 1982); T7 RNA polymerase promoters; T3 RNA polymerasepromoters; SP6 RNA polymerase promoters and other promoters effective inthe host cell of interest. Pol I promoters for expression of uncappedRNA are ubiquitous in all eukaryotes and include human RNA polymerase I(see Molecular Cell Biology, Darnell et al. eds 1986, pp. 311, 365-6).RNA polymerase III promoters may also be used in the present invention.

A coding sequence is “under the control of”, “functionally associatedwith” or “operatively associated with” transcriptional and translationalcontrol sequences (e.g., a pol I or pol II promoter) in a cell when RNApolymerase transcribes the coding sequence into RNA, e.g., mRNA or vRNA.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing an RNA (including cRNA, vRNA and virus mRNA) or protein byactivating the cellular functions involved in transcription andtranslation of a corresponding gene or DNA sequence. A DNA sequence isexpressed in or by a cell to form an “expression product” such as aprotein. The expression product itself, e.g. the resulting protein, mayalso be said to be “expressed” by the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell so that the host cell will express the introduced gene orsequence to produce a desired polypeptide, coded by the introduced geneor sequence. The introduced gene or sequence may also be called a“cloned” or “foreign” gene or sequence, may include regulatory orcontrol sequences, such as start, stop, promoter, signal, secretion, orother sequences used by a cell's genetic machinery. The gene or sequencemay include nonfunctional sequences or sequences with no known function.A host cell that receives and expresses introduced DNA or RNA has been“transformed” and is a “transformant” or a “clone.” The DNA or RNAintroduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or cells of a differentgenus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Plasmids are preferred vectors of the invention.

Vectors typically comprise the DNA of a transmissible agent, into whichforeign DNA is inserted. A common way to insert one segment of DNA intoanother segment of DNA involves the use of enzymes called restrictionenzymes that cleave DNA at specific sites (specific groups ofnucleotides) called restriction sites. A “cassette” refers to a DNAcoding sequence or segment of DNA that codes for an expression productthat can be inserted into a vector at defined restriction sites. Thecassette restriction sites are designed to ensure insertion of thecassette in the proper reading frame. Generally, foreign DNA is insertedat one or more restriction sites of the vector DNA, and then is carriedby the vector into a host cell along with the transmissible vector DNA.A segment or sequence of DNA having inserted or added DNA, such as anexpression vector, can also be called a “DNA construct.” A common typeof vector is a “plasmid”, which generally is a self-contained moleculeof double-stranded DNA, usually of bacterial origin, that can readilyaccept additional (foreign) DNA and which can readily introduced into asuitable host cell. A plasmid vector often contains coding DNA andpromoter DNA and has one or more restriction sites suitable forinserting foreign DNA. Coding DNA is a DNA sequence that encodes aparticular amino acid sequence for a particular protein or enzyme.Promoter DNA is a DNA sequence which initiates, regulates, or otherwisemediates or controls the expression of the coding DNA. Promoter DNA andcoding DNA may be from the same gene or from different genes, and may befrom the same or different organisms. Recombinant cloning vectors willoften include one or more replication systems for cloning or expression,one or more markers for selection in the host, e.g. antibioticresistance, and one or more expression cassettes.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, a gene encoding a polypeptide comprising a sequence from alibrary of sequences is heterologous to the vector DNA in which it isinserted for cloning or expression, and it is heterologous to a hostcell containing such a vector, in which it is expressed.

As noted above, the invention permits generation of reassortment virusesusing heterologous viral genes. In addition to incorporating genes forviral antigens in a genetic background of a virus strain adapted to growwell in culture, the invention permits creating cross-speciesreassortments, e.g., an influenza B antigen in an influenza Abackground.

Vaccines

As noted above, the present invention provides an efficient and economicstrategy for production of vaccines for treating or preventing RNA viralinfections, preferably, negative strand RNA virus infections. Theminimal plasmid-based system of the invention eliminates the need forselection, and provides close control of reassortment viruses. For theproduction of an inactivated influenza vaccine six plasmids containingthe non glycoprotein segments (e.g., PB1, PB2, PA, NP, M and NS) from ahigh yield strain (e.g., PR/8/34 (H1N1)) can be co-transfected with twoexpression plasmids containing the HA and NA cDNA of the recommendedvaccine subtype. Since no helper virus is required, the generated virusis an influenza virus with the desired gene constellation. An analogousapproach may be used to produce any variety of inactivated, reassortmentRNA virus for use in a vaccine. Expression plasmids comprising viralgene segments for a target virus (e.g, nonglycoprotein segments) may becotransfected with other expression plasmids encoding proteinscorresponding to a given infectious viral subtype (e.g, a viral subtypewhich is currently circulating in the population). Virus produced inaccordance with the invention can be used in traditional or newapproaches to vaccination (see Bilsel and Kawaoka, In: Nicholson,Webster and May (eds.), Textbook of Influenza, Chapter 32, pp. 422-434),particularly in the development of live, attenuated vaccines (discussedin greater detail infra). In particular, the present invention overcomesdefects of current technology, with respect to development reassortmentviruses with limited host range or unpredictable attenuation (id).

Much efforts has gone into the development of influenza vaccines (seeWood and Williams, In: Nicholson, Webstern and May (eds.), Textbook ofInfluenza, Chapter 23, pp. 317-323). While much of this section relatesto influenza vaccines, the scope of the present invention extends to allRNA virus vaccines, preferably, negative strand segmented RNA virusvaccines and particularly to Orthomyxoviridae vaccines.

Three types of inactivated influenza vaccines are currently available:whole virus, split-product, and surface antigen vaccines (see Wood, In:Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 25,pp. 333-345). Because the present invention permits the rapiddevelopment of a desired reassortment virus with acceptable growthcharacteristics in culture, it advantageously positions a vaccinemanufacturer to generate a sufficient quantity of vaccine to meet publichealth needs and ensure standardization, which is an importantrequirement currently mitigated by the need to produce clinicalquantities of vaccine, usually an 8 to 9 month period (Wood, supra, p.333).

Vaccine safety is also a concern (see Wiselka, In: Nicholson, Websterand May (eds.), Textbook of Influenza, Chapter 26, pp. 346-357). Becausethe vaccines of the invention permit production in defined cell culturesystems, they avoid non-specific pathogens, bacteria, and allergenicproteins that may be present in commercial vaccines prepared inembryonated eggs.

Adjuvants have been used with vaccines (e.g, influenza vaccines) (Woodand Williams, supra). The term “adjuvant” refers to a compound ormixture that enhances the immune response to an antigen. An adjuvant canserve as a tissue depot that slowly releases the antigen and also as alymphoid system activator that non-specifically enhances the immuneresponse (Hood, et al., Immunology, Second Ed., 1984, Benjamin/Cummings:Menlo Park, Calif., p. 384). Often, a primary challenge with an antigenalone, in the absence of an adjuvant, will fail to elicit a humoral orcellular immune response. Adjuvants include, but are not limited to,complete Freund's adjuvant, incomplete Freund's adjuvant, saponin,mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil orhydrocarbon emulsions, and potentially useful human adjuvants such asBCG (bacille Calmette-Guerin) and Corynebacterium parvum. An example ofa preferred synthetic adjuvant is QS-21. Alternatively, or in addition,immunostimulatory proteins, as described below, can be provided as anadjuvant or to increase the immune response to a vaccine. Preferably,the adjuvant is pharmaceutically acceptable.

The phrase “pharmaceutically acceptable” refers to molecular-entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Sterile water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin.

Vaccination effectiveness may be enhanced by co-administration of animmunostimulatory molecule (Salgaller and Lodge, J. Surg. Oncol. 1998,68:122), such as an immunostimulatory, immunopotentiating, orpro-inflammatory cytokine, lymphokine, or chemokine with the vaccine,particularly with a vector vaccine. For example, cytokines or cytokinegenes such as interleukin IL-1, IL-2, IL-3, IL-4, IL-12, IL-13,granulocyte-macrophage (GM)-colony stimulating factor (CSF) and othercolony stimulating factors, macrophage inflammatory factor, Flt3 ligand(Lyman, Curr. Opin. Hematol., 5:192, 1998), as well as some keycostimulatory molecules or their genes (e.g., B7.1, B7.2) can be used.These immunostimulatory molecules can be delivered systemically orlocally as proteins or by expression of a vector that codes forexpression of the molecule.

Dendritic Cell Targeting. Vaccination may be accomplished through thetargeting of dendritic cells (Steinman, J. Lab. Clin. Med., 128:531,1996; Steinman, Exp. Hematol., 24:859, 1996; Taite et al., Leukemia,13:653, 1999; Avigan, Blood Rev., 13:51, 1999; DiNicola et al.,Cytokines Cell. Mol. Ther., 4:265, 1998). Dendritic cells play a crucialrole in the activation of T-cell dependent immunity. Proliferatingdendritic cells can be used to capture protein antigens in animmunogenic form in situ and then present these antigens in a form thatcan be recognized by and stimulates T cells (see, e.g., Steinman, Exper.Hematol. 24:859-862, 1996; Inaba, etal., J. Exp. Med., 188:2163-73, 1998and U.S. Pat. No. 5,851,756). For ex vivo stimulation, dendritic cellsare plated in culture dishes and exposed to (pulsed with) virions in asufficient amount and for a sufficient period of time to allow the viralantigens to bind to the dendritic cells. The pulsed cells can then betransplanted back to the subject undergoing treatment, e.g., byintravenous injection. Preferably autologous dendritic cells, i.e.,dendritic cells obtained from the subject undergoing treatment, areused, although it may be possible to use MHC-Class II-matched dendriticcells, which may be obtained from a type-matched donor or by geneticengineering of dendritic cells to express the desired MHC molecules (andpreferably suppress expression of undesirable MHC molecules.)

Preferably, the dendritic cells are specifically targeted in vivo foruptake of virus or viral subunits. Various strategies are available fortargeting dendritic cells in vivo by taking advantage of receptors thatmediate antigen presentation, such as DEC-205 (Swiggard et al., Cell.Immunol., 165:302-11, 1995; Steinman, Exp. Hematol., 24:859, 1996) andFc receptors.

Inactivated Vaccines

Inactivated virus vaccines are well established for vaccinating againstRNA virual infection (e.g, influenza) (see Nichol, In: Nicholson,Webster and May (eds.), Textbook of Influenza, Chapter 27, pp. 358-372).To prepare inactivated virus, the transfected virus is grown either incell culture or in embryonated eggs. Virus can be inactivated treatmentwith formaldehyde, beta-propiolactone, ether, ether with detergent (suchas Tween-80), cetyl trimethyl ammonium bromide (CTAB) and Triton N101,sodium deoxycholate and tri(n-butyl) phosphate (Furminger, supra; Woodand Williams, supra). Inactivation can occur after or prior toclarification of allantoic fluid (from virus produced in eggs); thevirions are isolated and purified by centrifugation (Furminger, supra,see p. 326). To assess the potency of the vaccine, the single radialimmunodiffusion (SRD) test can be used (Schild et al., Bull. WorldHealth Organ. 1975, 52:43-50 and 223-31 Mostow et al., J. Clin.Microbiol. 1975, 2:531). The dose needed for a satisfactory immuneresponse has been standardized and is 15 μg HA/strain/dose. Theinactivated vaccine can be administered intramuscularly by injection.

Live Attenuated Influenza Vaccines

Attenuated cold adapted live RNA viral vaccines (influenza vaccines)have been developed (see Keitel and Piedra, In: Nicholson, Webster andMay (eds.), Textbook of Influenza, Chapter 28, pp. 373-390; Ghendon, In:Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 29,pp. 391-399). The ability to generate influenza virus entirely fromeight plasmids allows adjustment of the attenuation of a vaccine strainand enables development of a vaccine strain optimally suited for anytarget population (see, Bilsel and Kawaoka, supra). Because theinfluenza strains A/Ann Arbor/6/60 (H2N2), or for influenza B/AnnArbor/1/66, are used for preparation of live attenuated vaccinescurrently, one would insert each of the six cDNAs of the internal genes(PB2, PB1, PA, NP, M, NS) of the influenza into a plasmid such aspHW2000. Two plasmids containing the glycoproteins HA and NA of arelevant influenza strain would be constructed and co-transfected withthe six master plasmids encoding the non-glycosylated influenzaproteins.

It is expected that the genetic modification of the coding or noncodingregion of the internal genes improves the safety, infectivity,immunogenicity and protective efficacy of the vaccine, in addition topermitting development of attenuated virus.

The manipulation of the HA gene can also increase the safety of avaccine strain. For example, removal of basic amino acids found in theconnecting peptide of H5 or H7 glycoproteins of highly pathogenic avianinfluenza A viruses can increase the safety of the vaccine.

Mucosal Vaccination. Mucosal vaccine strategies are particularlyeffective for many pathogenic viruses, since infection often occurs viathe mucosa. The mucosa harbors dendritic cells, which are importanttargets for EBNA-1 vaccines and immunotherapy. Thus, mucosal vaccinationstrategies for inactivated and attenuated virus vaccines arecontemplated. While the mucosa can be targeted by local delivery of avaccine, various strategies have been employed to deliver immunogenicproteins to the mucosa.

In a specific embodiment, the vaccine can be administered in anadmixture with, or as a conjugate or chimeric fusion protein with,cholera toxin, such as cholera toxin B or a cholera toxin A/B chimera(Hajishengallis , J Immunol., 154:4322-32, 1995; Jobling and Holmes,Infect Immun., 60:4915-24, 1992). Mucosal vaccines based on use of thecholera toxin B subunit have been described (Lebens and Holmgren, DevBiol Stand 82:215-27, 1994). In another embodiment, an admixture withheat labile enterotoxin (LT) can be prepared for mucosal vaccination.

Other mucosal immunization strategies include encapsulating the virus inmicrocapsules (U.S. Pat. No. 5,075,109, No. 5,820,883, and No.5,853,763) and using an immunopotentiating membranous carrier (WO98/0558). Immunogenicity of orally administered immunogens can beenhanced by using red blood cells (rbc) or rbc ghosts (U.S. Pat. No.5,643,577), or by using blue tongue antigen (U.S. Pat. No. 5,690,938).

EXAMPLES

The present invention will be better understood by reference to thefollowing examples, which illustrate the invention without limiting it.

Example 1 “Ambisense” Approach for the Generation of Influenza A Virus:vRNA and mRNA Synthesis from One Template

As a first step in reducing the number of plasmids, this Example reportsthe construction and transfection of plasmids containing both the pol Iand pol II-promoter on the same plasmid and presents evidence that thissystem allows the expression of vRNA and protein from one template. ThisExample has been published (Hoffmann et al., Virology 2000, 267:310).

Materials and Methods

Cloning of plasmids. All cloning and PCR reactions were performedaccording to standard protocols. Briefly, the expression plasmids forthe polymerase complex genes of A/WSN/33 were derived from pcDNA3(Invitrogen) containing the immediate early promoter of the humancytomegalovirus (CMV) and the poly A site of the gene encoding bovinegrowth hormone (BGH). The viral cDNAs were derived from the plasmidspWNP 143, pWSNPA3, pWSNPB2-14, pGW-PB1 to yield the expressionconstructs pHW25-NP, pHW23-PA, pHW21-PB2, pHW22-PB1. pHW12 was generatedby inserting human pol I promoter and terminator sequences between thepol II-promoter and the polyA-site. The plasmid pHW52 was derived frompHW12 by first inserting oligonucleotides containing the noncodingregion of PB1 extended by HindIII and XhoI sites and then inserting thePB1-coding region from pHW22-PB1 into these sites. The plasmid pHW82-PB1was derived from pHW52-PB1 by deletion of the CMV-promoter sequences.The coding region for the enhanced green fluorescent protein (EGFP) inthe reporter construct pHW72-EGFP was obtained after PCR-amplificationusing pEGFP-N1 (Clontech) as template and inserting the cDNA afterSacII/XhoI digestion into the plasmid pHW72 containing the human pol Ipromoter and murine terminator and the noncoding region of the M-segmentseparated by SacII/Xhol sites. pHW127-M and pHW128-NS were constructedby RT-PCR amplification of viral RNA with the primers containing segmentspecific sequences and BsmBI sites for insertion into the BsmBI digestedvector pHH21 (E. Hoffmann, Ph.D. Thesis 1997, Justus Liebig University,Giessen, Germany; Neumann et al., Proc. Natl. Acad. Sci. USA 1999,96:9345). The construction of the plasmids pPolI-WSN-PB1; pPolI-WSN-PB2,pPolI-WSN-PA, pPolI-WSN-NP, pPolI-WSN-HA, pPolI-WSN-NA, pEWSN-HA, andpCAGGS-WNA15 has been described elsewhere (Neumann et al., supra).

Cell culture and transfection. Madin-Darby canine kidney (MDCK) cellswere maintained in modified Eagle-Medium (MEM) containing 10% fetalbovine serum (FBS), 293T human embryonic kidney cells were cultured inDulbecco's modified Eagle medium (DMEM) containing 10% FBS. TransIT LT-1(Panvera, Madison, Wis.) was used according to the manufacturer'sinstructions to transfect 1×106 293T cells. Different amounts ofplasmids (Table 1) were mixed with TransIT LT-1 (2 μl TransIT LT-1 per 1μg of DNA), incubated at room temperature for 45 min and added to thecells. Six hours later, the DNA-transfection mixture was replaced byOpti-MEM (Gibco/BRL, Gaithersburg, Md.) containing 0.3% bovine serumalbumin (BSA) and 0.01% FBS. Forty-eight hours after transfection,supernatants containing virus were titrated in MDCK cells.

RNA isolation and RT-PCR. Viral RNA was isolated from virus particleswith the use of the RNeasy-Kit (Qiagen, Valencia, Calif.) according tothe manufacturer's instructions. For characterization of recombinantinfluenza viruses the Access RT-PCR kit (Promega, Madison, Wis.) wasused according to the protocol provided. The following primers were usedin the RT-PCR experiments: Seq-PB1#1: 5′-AGG ATG GGA TTC CTC AAG G-3′(SEQ ID NO:1); Seq-PB1#2: 5′-GCT ATG GTT TCC AGA GCC CG-3′ (SEQ IDNO:2); Bm-PB1-1: 5′-TAT TCG TCT CAG GGA GCG AAA GCA GGC A-3′ (SEQ IDNO:3); Bm-PB1-2341R: 5′-ATA TCG TCT CGT ATT AGT AGA AAC AAG GCA TTT-3′(SEQ ID NO:4). RT-PCR experiments were performed by using the PTC-200DNA engine (MJ Research, Watertown, Mass.). The amplification programstarted with 1 cycle at 48° C. for 45 min (first-strand cDNA synthesis),and 1 cycle at 94° C. for 2 min (inactivation of the AMV reversetranscriptase and cDNA denaturation). These cycles were followed by 40cycles at 94° C. for 20 sec, 52° C. for 30 sec, and 72° C. for 30 sec(PCR amplification); the program ended with one cycle at 72° C. for 5min. The PCR products were analyzed by agarose gel electrophoresis andsequenced with the primer Seq-PB1#1 or Seq-PB1#2.

Flow cytometry. Forty-eight hours after transfection, 293T cells werewashed with phosphate-buffered saline (PBS), pelleted, and resuspendedin PBS plus 5% FBS. Flow cytometric analysis was performed by using aFACS Calibur flow cytometer (Becton Dickinson) and the data wereanalyzed by using the CellQuest software package. For EGFP expressionanalysis we used the emission wavelength of 530 nm (FL1) to achieve ahigh sensivity for EGFP mediated fluorescence detection.

Results and Discussion

Design and features of the cloning vector pHW12 containing twoeukaryotic promoters. Influenza A viruses are segmented viruses thatcontain RNA molecules with negative sense polarity. During thereplication cycle, recognition of the 5′- and 3′-structures of the eightvRNA segments by the ribonucleoprotein complex proteins (PB2, PB1, PA,NP) results in the replication and transcription of the influenza virusgenes. The fact that the terminal sequence elements are highly conservedindicates that a transcribed artificial RNA should have sequences thatare the same as those of the 5′- and 3′-ends (Luo et al., J. Virol.1991, 65:2861; Flick et al., RNA 1996, 2:1046). The cloning vector pHW12was constructed, allowing the insertion of sequences of interest betweenthe pol I promoter and terminator by using the restriction endonucleaseBsmBI. The pol I transcription unit is flanked by the pol II promoterfrom the cytomegalovirus (CMV) and by the polyadenylation signal of thegene encoding bovine growth hormone. The CMV-promoter, the poly A site,and the backbone of the plasmid are derived from the cloning vectorpcDNA3.

PB1 protein expression in the pol I/pol II bidirectional transcriptionsystem. To test the pol I/pol II one plasmid transcription system, cDNAof the PB1 gene of A/WSN/33 virus was inserted into the cloning vectorpHW12 to yield the plasmid pHW52-PB1. HindIII and XhoI restriction siteswere inserted into the 5′ and 3′ noncoding regions of this gene. Thesegenetic tags were included to ensure that the generated recombinantvirus could be identified by RT-PCR. We expected that human cellstransfected with this plasmid would yield two types of RNA: PB1-vRNAsynthesized by cellular pol I and an mRNA with a 5′-cap structuresynthesized by the pol II. Translation of the mRNA should result in thesynthesis of PB1-protein.

To examine whether the PB1-protein is produced from this construct, wetested replication and transcription of an artificial vRNA byconstructing the expression plasmids pHW21-PB2, pHW23-PA, and pHW25-NP,which contain cDNAs encoding PB2, PA and NP proteins of A/WSN/33 underthe control of the CMV-promoter. For the in vivo synthesis of anartificial vRNA, we constructed the reporter plasmid pHW72-EGFP,containing the EGFP cDNA flanked by the noncoding region of theM-segment and the human pol I-promoter and the murine terminatorsequence. Five plasmids (2 μg pHW21-PB2, 2 μg pHW52-PB1 (pol I/pol IIpromoter construct), 2 μg pHW23-PA, 2 μg pHW25-NP, and 1 μg pHW72-EGFP)were transfected into 293T cells. Twenty-four and 48 h aftertransfection, the cells were analysed by fluorescence microscopy. After24 hours fluorescent cells were observed. This result shows that within24 hours the polymerase proteins are synthesized in a concentrationsufficient to allow recognition of the influenza virus specific ends ofthe EGFP-vRNA. These proteins synthesize mRNA which is translated intoEGFP.

To evaluate the efficiency of this system, we performed flow cytometricanalysis to count the number of fluorescent cells. Forty-eight hoursafter transfection of the five plasmids, 18.72% of the cell populationshowed fluorescence. Only a background level of fluorescent cells(0.06%) was observed when pHW52-PB1 plasmid was not added; this findingis consistent with those of earlier studies showing that all four RNPcomplex proteins are necessary for the amplification of the vRNA (Huanget al., J. Virol. 1990, 64:5669). The results indicate that the PB1-cDNAtranscription and the resulting concentration of PB1 protein togetherwith the other RNP complex proteins is sufficient to initiate a viraltranscription/replication process.

Generation of recombinant influenza A virus. For the generation ofinfectious influenza A virus, it is necessary that the plasmid pHW52-PB1provides not only PB1 mRNA and protein but also sufficient amounts ofPB1-vRNA, which can be packaged into progeny virus. For the remainingseven vRNAs, we used plasmids that contain the cDNAs for the full-lengthRNAs of the A/WSN/33 virus, flanked by the human pol I promoter and themurine terminator. Transfection of these plasmids should result in thesynthesis of all eight viral RNAs that are replicated and transcribed bythe polymerase proteins forming new vRNPs. After synthesis of thestructural proteins, the RNPs would be packaged into new virusparticles.

We transfected 293T cells with different amounts of pHW52-PB1 plasmid(0, 2, 4 μg) together with the plasmids pPolI-WSN-PB2, pPolI-WSN-PA,pPolI-WSN-HA, pPolI-WSN-NP, pPolI-WSN-NA, pHW127-M, pHW128-NS (1 μgeach). The protein expression plasmids pHW21-PB2 (1 μg), pHW23-PA (0.1μg), pHW25-NP (1 μg), pEWSN-HA (1 μg), and pCAGGS-WNA15 (1 μg) werecotransfected. The expression plasmids for the hemagglutinin (HA) andthe neuraminidase (NA) were included to increase the yield oftransfectant virus.

Forty-eight hours after transfection, the supernatant of the primarytransfected 293T cells was transferred to MDCK cells. In alltransfection experiments in which pHW52-PB1 plasmid was added, 24 hoursafter the passage we observed a virus-induced cytopathic effect. Nocytopathic effect was visible if no PB1-expressing plasmid was includedin the transfection reaction. The virus titer was determined bytitrating the supernatant of the transfected cells on MDCK cells; thesupernatant was found to contain 2×10⁴-2×10⁵ pfu/ml. This finding showsthat after transfection of the PB1-pol I/pol II-promoter plasmid(together with the expression plasmids) PB1 vRNA and PB1 protein aresynthesized in the human cell line 293T at a level sufficient for thegeneration of infectious influenza A viruses. In the cotransfectionexperiments with plasmids containing the PB1-cDNA separated on twoplasmids (pHW82-PB1 and pHW22-PB1), a virus titer of 2×10⁴ pfu/ml wasfound; the analogous experiment using the plasmids with wild-type PB1sequences (pPol I-WSN-PB1 and pHW22-PB1) resulted in a virus titer of3×10⁶ pfu/ml.

Unlike the expression construct with a pol II-promoter used in aprevious study (Neumann et al., Proc. Natl. Acad. Sci. USA 1999,96:9345), we used the plasmid pHW52-PB1 that contains sequences derivedfrom the pol I-transcription unit that are inserted between theCMV-promoter and the polyadenylation site. The expression of the EGFPreporter gene demonstrates that the overall expression of PB1-protein inthis system is sufficient for formation of EGFP-RNP complexes. Althoughthe pol I-promoter/terminator region contains recognition sequences forpol I specific transcription and termination factors (Beckmann et al.,Science 1995, 270: 1506; Bell et al., Science 1988, 241:1192; Kuhn etal, EMBO J. 1994, 13:416), these DNA binding proteins do not seem toinhibit pol II-mediated transcription. These findings are consistentwith the finding that the pol I-specific DNA binding proteins are moreabundant in the nucleolus, the compartment in which the cellularrDNA-transcription takes place (Evers et al., EMBO J. 1995, 14:1248).These results indicate that after transfection of the pol I/polII-promoter construct into the cell, some of the plasmids are deliveredto the nucleolus, where the pol I-mediated transcription occurs and someare retained in the nucleus, where they are transcribed by RNApolymerase II.

Because the reporter construct pHW52-PB1 contained additionalnon-influenza virus sequences (restriction sites) in the noncodingregion before the start codon and after the stop codon, we wereinterested whether these sequences were stably maintained in the viralPB1 RNA segment. Therefore, we isolated vRNA after the second passage oftransfectant virus on MDCK cells and performed reverse transcription-PCRanalysis. The amplification of vRNA with PB1-specific primers resultedin the generation of cDNA-fragments of the expected sizes. With the sameviral RNA and primers, but without the addition of reversetranscriptase, no amplification product was obtained, showing that thecDNA originated from viral RNA and not from plasmid DNA carried overfrom the supernatant of transfected cells.

Sequencing of the PCR-products revealed that both restriction sitesequences were present in the RNA molecule. The results show that thepol I/pol II transcription system allows recovery of infectiousrecombinant virus and that virus with foreign sequences in the noncodingregion of the PB1 gene is viable. This modified PB1-segment is stillreplicated, transcribed, and packaged into virus particles. Previously,by using the RNP transfection system the noncoding region of influenza Avirus segments were changed. By substituting the noncoding region of theNA gene with the corresponding sequence of the NS-segment of influenza Btransfectant influenza viruses were obtained (Muster et al., Proc. Natl.Acad. Sci. USA 1991, 88:5177; Bergmann and Muster, J. Gen. Virol. 1995,76:3211). This type of virus with a chimeric NA segment showed anattenuated phenotype in mice and protected mice inoculated with a nonlethal dose against infection of the wild-type influenza virusinfection. These results showed that the genetic alteration of thenoncoding region of an RNA segment can change the biologic property of atransfectant virus. Here, we report for the first time that evennon-influenza virus sequences can be inserted into the noncoding regionof the PB1 segment.

With the pol I/pol II transcription system it is now possible tosystematically modify these sequence elements in the noncoding region ofthe PB1 segment and to evaluate whether these genetic manipulationsresult in changes in the biologic properties of the recombinant viruses.Indeed, the lower yield of the viruses with the mutated PB1 segmentcompared to the wild-type virus indicates that the inserted sequencesnegatively influence the virus growth.

Although the plasmid-based system developed recently (Neumann et al.,supra) is highly efficient in generating influenza virus, it involvescloning 14-17 plasmids. In this study we reduced the number of plasmidsto 13 needed for the efficient recovery of influenza A/WSN/33 virusstrain. The reduction in the number of plasmids achieved by thisapproach promises to increase the efficiency of transfection for celllines other then 293T cells, thus allowing the delivery of genes to celllines for which the efficient delivery of 14 plasmids is difficult toachieve. Fodor et al. (J. Virol. 1999, 73:9679) were able to rescueinfluenza virus after transfecting 12 plasmids, but the virus yield inthat study was only 1-2 infectious virus particles per 10⁶ transfectedVero cells.

Example 2 Construction of Recombinant Influenza A Viruses from a MinimalPlasmid-Based System

This example describes use of the plasmid-based transfection system forthe rescue of influenza A virus entirely from cloned cDNA. Unlikeestablished plasmid-based systems, this system for the generation ofinfluenza A virus employs the construction and transfection of onlyeight expression plasmids, each containing one copy of a different viralcDNA corresponding to a viral gene segment. This reverse-genetics systemreduces the number of plasmids required for the recovery of influenza Aviruses and allows the predictable and efficient generation ofreassortment viruses.

Materials and Methods

Cloning of plasmids. The plasmid pHW2000 (FIG. 3A) was derived frompHW12 (Example 1). The pHW2000 cloning vector contains 225 bp of thehuman pol I promoter and 33 bp of the murine terminator sequenceseparated by two BsmBI sites. The pol I promoter and terminator elementsare flanked by a truncated immediate-early promoter of the humancytomegalovirus (starting approximately 350 bp upstream of thetranscription start site as found in pcDNA3, Invitrogen, Carlsband,Calif.) and by the polyadenylation signal of the gene encoding bovinegrowth hormone. The eight plasmids containing the cDNA of the virusA/WSN/33 (H1N1) (pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA,pHW185-NP, pHW186-NA, pHW187-M, and pHW188-NS) were constructed byinserting ApaI-SalI fragments (with viral cDNA and pol I promoter andterminator sequences) of the plasmids pPolI-WSN-PB2, pPolI-WSN-PB1,pPolI-WSN-PA, pPolI-WSN-NP, pPolI-WSN-HA, pPolI-WSN-NA (Neumann et al.,Proc. Natl. Acad. Sci. USA 1999, 96:9345), pHW127-M, and pHW128-NS(Example 1) into the Apal-SalI vector fragment of pHW2000. The eightplasmids containing the cDNA of A/Teal/HK/W312/97 (H6N1) (pHW241-PB2,pHW242-PB1, pHW243-PA, pHW244-HA, pHW245-NP, pHW246-NA, pHW247-M, andpHW248-NS) were constructed by reverse-transcriptase polymerase chainreaction (RT-PCR) amplification of the viral RNA. The primers used inthe PCR reaction contained segment-specific sequences at their 3′ endand BsmBI or BsaI restriction site sequences at their 5′ end. Afterdigestion of the PCR products with BsmBI or BsaI, the fragments werecloned into the vector pHW2000 (FIG. 3A). The sequences of the primersused for amplification of the genome of A/teal/HK/W312/97 (H6N1) follow.The primers are shown from left to right corresponding to the 5′ and 3′ends. The influenza A specific nucleotides are underlined. NS: Bm-NS#1:(SEQ ID NO:5) TATTCGTCTCAGGGAGCAAAAGCAGGGTG Bm-NS#2: (SEQ ID NO:6)ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTT M: Bm-M#1: (SEQ ID NO:7)TATTCGTCTCAGGGAGCAAAAGCAGGTAG Bm-M#2: (SEQ ID NO:8)ATATCGTCTCGTATTAGTAGAAACAAGGTAGTTTTTT NA: Bm-NA1-1: (SEQ ID NO:9)TATTCGTCTCAGGGAGCAAAAGCAGGAGTTTAACATG Bm-NA-1413R: (SEQ ID NO:10)ATATCGTCTCGTATTAGTAGAAACAAGGAGTTTTT HA: Bm-H6-1: (SEQ ID NO:11)TATTCGTCTCAGGGAGCAAAAGCAGGGGAAAATG Bm-NS#2: (SEQ ID NO:6)ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTT (note: HA and NS segment have theidentical sequence in this part of the noncoding region) NP: Ba-NP-1:(SEQ ID NO:12) TATTGGTCTCAGGGAGCGAAAGCAGGGTA Ba-NP1565R: (SEQ ID NO:13)ATATGGTCTCGTATTAGTAGAAACAAGGGTATT PA: Bm-PA1-1: (SEQ ID NO:14)TATTCGTCTCAGGGAGCGAAAGCAGGTACTGATCC Bm-PA1-2231R: (SEQ ID NO:15)ATATCGTCTCGTATTAGTAGAAACAAGGTACTTTTT PB1: Bm-PB1a-1: (SEQ ID NO:16)TATTCGTCTCAGGGAGCGAAAGCAGGCAAACC Bm-PB1-2341R: (SEQ ID NO:4)ATATCGTCTCGTATTAGTAGAAACAAGGCATTT PB2: Ba-PB2-1: (SEQ ID NO:17)TATTGGTCTCAGGGAGCGAAAGCAGGTCAATTATATTC Ba-PB2-2341R: (SEQ ID NO:18)ATATGGTCTCGTATTAGTAGAAACAAGGTCGTTTTTThe RT-reaction was performed with the primer 5′-AGCAAAAGCAGG-3′ (SEQ IDNO:19). To ensure that the viral cDNAs derived from RT-PCR amplificationin the expression plasmids did not have unwanted mutations, the insertedcDNAs were sequenced.

Viruses and cell culture. Influenza viruses A/WSN/33 (H1N1) andA/Teal/HK/W312/97 (H6N1) were propagated in 10-day-old eggs. Madin-Darbycanine kidney (MDCK) cells were maintained in modified Eagle Medium(MEM) containing 10% FBS. 293T human embryonic kidney cells werecultured in Opti-MEM I (Life Technologies, Gaithersburg, Md.) containing5% FBS. For the transfection experiments six well tissue culture plateswere used. The day before transfection confluent 293T and MDCK cells ina 75 cm² flask were trypsinized and 10% of each cell line was mixed in18 ml OptiMEM I; 3 ml of this cell suspension was seeded into one wellof a six well plate. The cocultured MDCK and 293T cells (0.2-1×10⁶ cellsper well each) were used for the transfection experiments. TransIT LT-1(Panvera, Madison, Wis.) was used according to the manufacturer'sinstructions to transfect the cells. Briefly, 2 μl of TransIT LT-1 per 1μg of DNA was mixed, incubated at room temperature for 45 min, and addedto the cells. Six hours later, the DNA-transfection mixture was replacedby Opti-MEM I. Thirty hours after transfection, 1 ml of Opti-MEM Icontaining TPCK-trypsin was added to the cells; this addition resultedin a final concentration of TPCK-trypsin of 0.5 μg/ml in the cellsupernatant. The virus titer of the cell supernatant was determined bytitration of the supernatant on MDCK cells.

RNA isolation and RT-PCR. Viral RNA was isolated from virus particleswith the RNeasy-Kit (Qiagen, Valencia, Calif.), which was used accordingto the manufacturer's instructions. For characterization of recombinantinfluenza viruses, the Access RT-PCR kit (Promega, Madison, Wis.) wasused according to the protocol provided. The following primers were usedin the RT-PCR experiments: Bm-NS#1 (5′-TAT TCG TCT CAG GGA GCA AAA GCAGGG TG-3; SEQ ID NO:5) and Bm-NS#2 (5′-ATA TCG TCT CGT ATT AGT AGA AACAAG GGT GTT TT-3; SEQ ID NO:6). RT-PCR experiments were performed byusing the PTC-200 DNA engine (MJ Research, Watertown, Mass.). Theamplification program started with 1 cycle at 48° C. for 45 min and 1cycle at 94° C. for 2 min. These cycles were followed by 40 cycles at94° C. for 20 sec. 52° C. for 30 sec. and 72° C. for 40 sec; the programended with one cycle at 72° C. for 5 min. The PCR products were analyzedby agarose gel electrophoresis and sequenced with the primer Bm-NS#1.The Center for Biotechnology at St. Jude Children's Research Hospitaldetermined the sequence of template DNA by using rhodamine or dRhodaminedye-terminator cycle sequencing ready reaction kits with AmpliTaq® DNApolymerase FS (Perkin-Elmer, Applied Biosystems, Inc. [PE/ABI], FosterCity, Calif.) and synthetic oligonucleotides. Samples were subjected toelectrophoresis, detection, and analysis on PE/ABI model 373, model 373Stretch, or model 377 DNA sequencers.

Results

Establishment of the pol I-pol II system for the generation of A/WSN/33(H1N1). Because the genome of influenza A virus contains eight segments,it was reasoned that the insertion of all eight influenza A cDNAsbetween a pol I promoter and a pol II promoter should result in thetranscription of the eight vRNAs, all viral in RNAs, and in thesynthesis of all 10 viral proteins (FIG. 1). After assembly of all viralribonucleoproteins with the structural proteins, infectious influenza Avirus should then be formed (FIG. 2).

To test whether infectious influenza A virus could be rescued with thiscDNA-bidirectional transcription system, the eight expression plasmids(pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA, pHW185-NP, pHW186-NA,pHW187-M, and pHW188-NS) were constructed containing the eight cDNAs ofA/WSN/33 (H1N1). Eight plasmids (1 μg of each plasmid) (Table 1) werecotransfected into transiently cocultured 293T-MDCK cells. Both celllines were cocultured in one cell-culture well the day beforetransfection to ensure conditions for high DNA transfection efficiency(293T cells) and for replication efficiency (MDCK cells) of influenza Aviruses. After 48 and 72 hours, the MDCK cells showed a virus-inducedcytopathic effect, but no cytopathic effect was observed aftertransfection of seven plasmids without the PB1-expression construct(Table 1). The virus titer of the supernatant was determined atdifferent times posttransfection by titration in MDCK cells. Twenty-fourhours posttransfection cell supernatant contained 1×10³ viruses per ml;2×10⁷ infectious viruses were generated 72 hours posttransfection(Table 1) per ml. The recovered viruses were passaged two times on MDCKcells. To verify that the generated virus was the designed A/WSN-virus,the cDNA was produced for the NS gene by RT-PCR (FIG. 4A, lane 8). Thegeneration of two fragments after digestion with the restrictionendonuclease NcoI (FIG. 4B, lane 8) and sequence analysis of theamplified fragment confirmed that the recovered virus was indeed thedesigned A/WSN virus. These findings show that the pol I and polII-driven synthesis of vRNA and mRNA from eight templates results in thegeneration of infectious influenza A virus. TABLE 1 Plasmid sets usedfor the recovery of A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1) VirusesSeg- ment A/WSN/33 (H1N1) A/Teal/HK/W312/97 (H6N1) 1 pHW181-PB2pHW181-PB2 pHW241-PB2 pHW241-PB2 2 — pHW182-PB1 — pHW242-PB1 3 pHW183-PApHW183-PA pHW243-PA pHW243-PA 4 pHW184-HA pHW184-HA pHW244-HA pHW244-HA5 pHW185-NP pHW185-NP pHW245-NP pHW245-NP 6 pHW186-NA pHW186-NApHW246-NA pHW246-NA 7 pHW187-M pHW187-M pHW247-M pHW247-M 8 pHW188-NSpHW188-NS pHW248-NS pHW248-NS virus titers§ t = 24 h 0 1 × 10³ 0 0 t =48 h 0  2 × 10⁶* 0 2 × 10³ t = 72 h 0  2 × 10⁷* 0  2 × 10⁵*§The numbers represent infectious virus particles per ml of thesupernatant of transfected cells as determined 24 h, 48 h and 72 h aftertransfection.*Cytopathic effect in the cocultured MDCK cells was observed.

Recovery of A/Teal/HK/W312/97 (H6N1) by cotransfecting eight plasmids.The influenza virus A/WSN/33 (H1N1), originally derived from the humaninfluenza pandemic strain from 1918 (Goto and Kawaoka, Proc. Acad. Sci.USA 1998, 95:10224; Reid et al., Proc. Natl. Acad. Sci. USA 1999,96:1651), has been passaged in mouse brain and is well adapted forgrowth in cell culture. To evaluate the efficiency of the eight-plasmidsystem for the generation of a virus from cloned cDNA that is notalready adapted for growth in cell culture, generation of the virusA/Teal/HK/W312/97 (H6N1) was attempted from cloned cDNA alone. This H6N1virus was isolated from a dead teal during the H5N1 outbreak in HongKong in 1997. Genetic analysis of this virus revealed that it has sevensegments with more than 97% nucleotide homology to the pathogenic H5N1virus strains. RNA was extracted from infected allantoic fluid, and theRT-PCR-amplified cDNAs were inserted into pHW2000; this insertionresulted in eight expression plasmids (FIG. 3). Seventy-two hours aftertransfection of pHW241-PB2, pHW242-PB1, pHW243-PA, pHW244-HA, pHW245-NP,pHW246-NA, pHW247-M, and pHW248-NS (1 μg each) into cocultured 293T-MDCKcells, a virus-induced cytopathic effect was observed in MDCK cells(Table 1). The virus yield was 2×10⁵ infectious viruses per ml of thesupernatant of the transfected cells. As shown in FIG. 4 (A and B, lane2), the identity of the recovered virus was verified by characterizationof the NS segment. These results illustrate that this plasmid systemrequires the cloning of only eight cDNAs into one plasmid vector andthat the transfection of the eight expression plasmids allows therecovery of an influenza A virus with the antigenicity of a virus notalready adapted to growth in mammalian cells.

Rescue of reassortment influenza A viruses. The utility of theeight-plasmid system was tested for the generation of reassortmentviruses. Because this DNA transfection system does not require anyselection system, the recovery of reassortment viruses should beachievable by appropriate combinations of expression plasmids in thetransfection reactions. Seven expression plasmids containing the cDNA ofA/Teal/HK/W312/97 (H6N1) were cotransfected with one expression plasmidcontaining the cDNA of A/WSN/33 (H1N1) (Table 2). High virus yields wereobtained for the reassortment viruses containing seven segments of theteal virus and the M segment or NS segment of the WSN virus. Lower virusyields were obtained for the NA and HA-reassortment viruses (Table 2).Because single reassortment viruses were rescued with the eight-plasmidsystem, the next step was to determine whether a virus could be rescuedwith multiple segments derived from one virus. Therefore, fourexpression plasmids containing the cDNA of the RNP-complex genes of theH6N1 virus (pHW241-PB2, pHW242-PB1, pHW243-PA and pHW245-NP) weretransfected together with the plasmids pHW184-HA, pHW186-NA, pHW187-M,and pHW188-NS containing the cDNA of the WSN virus (Table 2). 4×10⁶viruses were recovered per ml of cell supernatant. As shown in FIG. 4(lane 10), the amplified NS segment of the quadruple reassortment viruswas cleaved by NcoI; thus, the NS segment is derived from the WSN virus.These results show that the eight-plasmid transfection system allows therecovery of single and quadruple reassortment viruses. TABLE 2Generation of reassortment influenza A viruses between A/Teal/HK/W312(H6N1) and A/WSN/33 (H1N1) by cotransfecting eight plasmids. segment* HANA M NS HA-NA-M-NS 1 pHW241-PB2 pHW241-PB2 pHW241-PB2 pHW241-PB2pHW241-PB2 2 pHW242-PB1 pHW242-PB1 pHW242-PB1 pHW242-PB1 pHW242-PB1 3pHW243-PA pHW243-PA pHW243-PA pHW243-PA pHW243-PA 5 pHW245-NP pHW245-NPpHW245-NP pHW245-NP pHW245-NP 4 pHW184-HA pHW244-HA pHW244-HA pHW244-HApHW184-HA 6 pHW246-NA pHW186-NA pHW246-NA pHW246-NA pHW186-NA 7 pHW247-MpHW247-M pHW187-M pHW247-M pHW187-M 8 pHW248-NS pHW248-NS pHW248-NSpHW188-NS pHW188-NS virus titer§ 2 × 10² 2 × 10³ 2 × 10⁵ 2 × 10⁷ 4 × 10⁶*plasmids containing the cDNA of A/WSN/33 (H1N1) are shown in bold§The numbers represent infectious virus particles per ml of supernatantof transfected cells as determined 72 h after transfection.

Discussion

The ability to rescue influenza A virus after transfection of the eightexpression plasmids containing the cDNA of A/Teal/HK/W312/97 (H6N1) orA/WSN/33 (H1N1) proves that the pol I-pol II transcription systemprovides sufficient amounts of vRNA and viral proteins for the formationof infectious influenza A virus. Two types of mRNAs that differ in theirnoncoding regions are synthesized (FIG. 1). The mRNA type encoding allviral proteins is directly transcribed by pol II. In addition to theinfluenza virus sequences of the non coding regions (NCR), these mRNAscontain sequences from the pol I promoter and the murine terminatorregions. Importantly, the pol I-pol II expression system developedcontained only 33 bp of the murine terminator sequences. Previousstudies using the reporter genes chloramphenicol acetyltransferase (CAT)and green fluorescent protein (GFP) showed that sequences in the 174-bpterminator region reduced pol II-mediated expression of protein(Hoffmann, E., Ph.D. Thesis 1997, Justus Liebig University, Giessen,Germany). A second mRNA type is generated after the initiation of theviral replication and transcription process (FIG. 2). This mRNA issynthesized by the viral polymerase proteins and contains a 5 capstructure derived from cellular RNAs by cap snatching preceding theinfluenza virus noncoding sequences. The structural proteins translatedfrom both mRNAs associate with the RNP complexes to form new virusparticles. After the budding of transfectant viruses, the generatedvirus particles can then replicate in the 293T cells and in thecocultured MDCK cells.

Unlike the approaches discussed in the Background of the Invention,supra, the method of the instant invention deploys the eight cDNAs ineight plasmids that contained 225 bp of the pol I promoter sequences and33 bp of the terminator sequences. In the pol I-pol II system, all 10viral proteins are expressed from a truncated immediate-early promoterof the human cytomegalovirus. The fact that the expression of allstructural proteins with the 17-plasmid system (Neumann et al., Proc.Natl. Acad. Sci. USA 1999, 96:9345) and with the 8-plasmid system (thisstudy) resulted in a higher efficiency of virus recovery than didcotransfection of plasmids expressing the RNP complex proteins (Neumannet al., supra; Fodor et al., J. Virol. 1999, 73:9679) supports the ideathat the generation of infectious influenza A virus is enhanced byproviding the HA, NA, M1, M2, and NS2 proteins early after transfection.

The viral replication cycle involves a complex interaction between theviral proteins with each other and with cellular factors (Ludwig et al.,Virol. Immunol. 1999, 12:175). Thus, for the generation of infectiousvirus, the plasmid-driven synthesis of viral molecules should provideoptimal concentrations of viral proteins for the initiation of thereplication cycle and for the formation of virus-like particles.Although the eight-plasmid system proved to be efficient, it might bepossible to further increase the production of virus. It was shown thatthe ratio of transfected plasmids expressing the RNP complex proteinsand the expression of the M1 protein influences the transcriptaseactivity (Pleschka et al., J. Virol. 1996, 70:4188; Perez and Donis,Virology 1998, 249:52). The efficiency of the formation of virus-likeparticles also depends on the concentration of structural viral proteins(Mena et al., J. Virol. 1996, 70:5016; Gomez-Puertas et al., J. Gen.Virol., 1999, 80:1635; Neumann et al., J. Virol. 2000, 74:547). Theefficiency of the generation of infectious virus with the pol I-pol IIsystem might therefore be further increased by varying the plasmidconcentrations used in the transfection reaction or by using expressionplasmids with different pol II promoters. Because the splicingefficiency mediated by cellular factors influences the ability ofinfluenza A virus to replicate (Lau and Scholtissek, Virology 1995,212:225), the use of cell lines other than 293T may increase the virusyield for certain influenza A strains. The high virus yield of thequadruple reassortment (Table 2) is consistent with the finding that therapid replication of A/WSN/33 (H1N1) in cultured cells is mediated bythe HA, NA, and M segments (Goto and Kawaoka, Proc. Natl. Acad. Sci. USA1998, 95:10224; Schulman and Palese, J. Virol. 1977, 24:170; Yesudaetal., J. Virol. 1994, 68:8141).

The generation of viable reassortants (Table 2) between the avian H6N1virus and the human H1N1 virus indicates that this H6N1 virus canacquire gene segments from a distantly related virus. Genetic analysissuggested that the pathogenic H5N1 viruses were generated byreassortment (Xu et al., Virology 1999, 261:15). H5N1-like gene segmentsare found in the H6N1 and H9N2 subtypes (Guan et al., Proc. Natl. Acad.Sci. USA 1999, 96:9363), a finding indicating that these viruses mayhave been precursors of the pathogenic H5N1 viruses. Reassortment eventsthat could create new pathogenic influenza viruses are likely to occurin the future. However, the ability to generate and manipulate theseviruses by the simplified method developed in this study will helpresearchers better understand the biological properties of these newviruses and develop efficient vaccines to protect a population againstthem. The length of the time period between the emergence of a newpathogenic strain and the preparation of a vaccine is a crucial variablein the effectiveness of a vaccination program. The ability to generateviruses by cloning only eight plasmids reduces the time needed for thegeneration of potential vaccine candidates and improves existingreverse-genetics systems by simplifying virus creation and reducing theoverall cost of production of a vaccine.

The concept of introducing viral cDNA between a pol I promoter and a polII promoter into eukaryotic cells for the recovery of virus is alsoapplicable for the generation of other members of the familyOrthomyxoviridae. For influenza B virus, this strategy would require theconstruction and cotransfection of eight plasmids; for influenza C,seven; and for Thogotovirus, six. The in vivo transcription of 5′-cappedmRNA as well as vRNA from the same cDNA template may also simplifyplasmid-based systems for other RNA viruses or even facilitate theestablishment of pol I-pol II systems for viruses from other families(e.g. Arenaviridae, Bunyaviridae).

Example 3 RNA Pol I/Pol II System for the Generation of Influenza BVirus Entirely from Cloned cDNA

Influenza A and B viruses each contain eight segments of single strandedRNA with negative polarity (for review see Lamb and Krug,“Orthomyxoviridae: The viruses and their replication”; in Fields (Ed.),Virology; p1353-1395). Unlike influenza A, the eight segments ofinfluenza B encode 11 proteins. The three largest genes code for thecomponents of the RNA polymerase, PB1, PB2 and PA; segment 4 encodes thehaemagglutinin. Segment 5 encodes the nucleoprotein, the majorstructural component associated with viral RNA, segment 6 encodes theneuraminidase (NA) and the NB protein. Both proteins, NB and NA, aretranslated from overlapping reading frames of a biscistronic mRNA.Segment 7 of influenza B also encodes two proteins: BM1 and BM2. Thesmallest segment encodes two products: NS1 is translated from the fulllength RNA, while NS2 is translated from a spliced mRNA.

Construction of expression plasmids containing the cDNA of influenza Binvolves the same strategy as described for the generation of theinfluenza A virus A/teal/HK/W312/97 (H6N1). First RNA is isolated fromvirus particles obtained from infected allantoic fluid, e.g., B/Lee/40.Based on the conserved sequences of the noncoding region, primers forthe RT-PCR are prepared and used for the synthesis of cDNA. At the5′-end those primers contain sequences for the restriction endonucleasesBsmBI or BsaI. Digestion of the PCR products with BsmBI or Bsa I allowsthe insertion into the cloning vector pHW2000 (or pHW11) linearized withBsmBI. To ensure that the cDNAs in the plasmids do not have unwantedmutations due to errors made by the polymerase during PCR, theconstructs have to be sequenced.

Co-transfection of cocultured 293T -MDCK cells (or COS-1-MDCK) cells andthe addition of trypsin results in the generation of infectiousinfluenza B virus. The supernatants of transfected cells are thenpassaged onto new MDCK cells. The resultant virus titer can bedetermined by standard methods, e.g., the HA assay and plaque assay.RT-PCR performed with specific primers for each gene segment allows theamplification of the RNA from the recombinant influenza B virus.Sequencing of the products confirms that the generated virus is indeedthe desired influenza B virus.

Example 4 Eight-Plasmid Rescue System for Master Strain Influenza AVirus

To determine the commercial utility of this plasmid-based system for theproduction of vaccines, we generated the masterstrain A/PR/8134 (H1N1),currently used for production of inactivated vaccine, entirely fromcloned cDNAs as described in Example 2. The virus yield as determined byHA-assay after passage of the recombinant virus into eggs was as high asthe virus yield of the parental wildtype virus. These results prove thatthe generated recombinant virus has the same growth properties as theparental egg grown virus and indicate that the eight-plasmidtransfection method has the potential to improve currently used methodsfor the production of vaccine viruses.

Materials and Methods

Viruses and Transfection. The Influenza virus A/PR/8/34 (H1N1) wasobtained from the repository of St. Jude Childrens's Research Hospitaland propagated in 10-day-old embryonated chicken eggs. Madin-Darbycanine kidney (MDCK) cells were maintained in MEM containing 10% FBS.293T human embryonic kidney cells and Vero cells were cultured inOpti-MEM I (Life Technologies, Gaithersburg, Md.) containing 5% fetalbovine serum (FBS). For the transfection experiments, six-well tissueculture plates were used. The cocultured MDCK and 293T cells (0.21×10⁶each of cells per well) were used for the transfection experiments.TransIT LT-1 (Panvera, Madison, Wis.) was used according to themanufacturer's instructions to transfect the cells. Briefly, 2 μl ofTransIT LT-1 per 1 μg of DNA was mixed, incubated at room temperaturefor 45 min, and added to the cells. Six hours later, theDNA-transfection mixture was replaced by Opti-MEM I. Twenty four hoursafter transfection, 1 ml of Opti-MEM I containing TPCK-trypsin was addedto the cells; this addition resulted in a final concentration ofTPCK-trypsin of 0.5 μg/ml in the cell supernatant. The virus titer wasdetermined by passage of the cell supernatant on MDCK cells by plaqueassay.

RT-PCR and Construction of Plasmids. Viral RNA was extracted from 200 μlof virus containing allantoic fluid of embryonated egg using QiagenRNeasy Kit. Two-step RT-PCR was employed to amplify each of the viralgene segments. Briefly, the RNA was transcribed into cDNA using AMVreverse transcriptase (Roche Diagnostics, Germany) according to theprotocol provided and then the cDNA was amplified using Expand HighFidelity PCR system (Roche Diagnostics, Germany). The amplificationprogram started with 1 cycle at 94° C. for 2 min; followed by 30 cyclesat 94° C. for 20 seconds, 54° C. for 30 seconds, 72° C. for 3 min; theprogram ended with one cycle at 72° C. for 5 minutes. The primers usedcontained either sequences for BsaI or BsmBI to allow the preciseinsertion of the digested PCR-fragments into the cloning vector pHW2000(see Example 2).

For cloning of the HA, NP, NA, M, NS genes the PCR-fragments weredigested with BsmBI or BsaI and ligated into the cloning vector pHW2000.For cloning of the P-genes two (PB2, PA) or three (PB1) fragments wereisolated, digested and ligated into pHW2000-BsmBI. To ensure that thegenes were free of unwanted mutations, the PCR-derived fragments weresequenced. The eight plasmids containing the full length cDNA ofA/PR/8/34 (H1N1) were designated pHW191-PB2, pHW192-PB1, pHW193-PA,pHW194-HA, pHW195-NP, pHW196-NA, pHW197-M, and pHW198-NS. The Center forBiotechnology at St. Jude Children's Research Hospital determined thesequence of template DNA by using rhodamine or dRhodamine dye-terminatorcycle sequencing ready reaction kits with AmpliTaq® DNA polymerase FS(Perkin-Elmer, Applied Biosystems, Inc. [PE/ABI], Foster City, Calif.)and synthetic oligonucleotides. Samples were subjected toelectrophoresis, detection, and analysis on PE/ABI model 373, model 373Stretch, or model 377 DNA sequencers.

Results

To allow intracellular synthesis of virus-like vRNAs and mRNAs, we haveestablished the RNA pol I-pol II expression system (see Example 2). Inthis system viral cDNA is inserted between the human RNA polymerase I(pol I) promoter and a terminator sequences. This entire pol Itranscription unit is flanked by an RNA polymerase II (pol II) promoterand a poly(A) site. The orientation of the two transcription unitsallows the synthesis of negative-sense viral RNA and positive-sense mRNAfrom one viral cDNA template. This pol I-pol II system starts with theinitiation of transcription of the two cellular RNA polymerase enzymesfrom their own promoters, presumably in different compartments of thenucleus (see FIG. 1). Transfection of eight plasmids into 293T cellsresults in the interaction of all molecules derived from the cellularand viral transcription and translation machinery, ultimately generatinginfectious influenza A virus. This system proved to be very efficientfor the formation of the influenza viruses A/WSN/33 (H1N1) andA/Teal/HK/W312/97 (H6N1) (Example 2).

Since the current master strain for production of inactivated influenzavaccine is A/PR/8/34 (H1N1), we attempted to generate this virusentirely from cloned cDNA. The cDNAs representing the eight RNA-segmentswere inserted into the vector pHW2000. The resultant plasmids(pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW196-NA,pHW197-M, and pHW198-NS) were transfected into cocultured 293T-MDCK orVero-MDCK cells. Seventy-two hours after transfection the virus titerwas determined by titration in MDCK cells. The supernatant of coculturedVero-MDCK cells contained 1×10⁴ pfu and the supernatant of cocultured293T-MDCK cells contained 2×10⁶ pfu per ml. The higher yield in293T-MDCK cells is most likely caused by the higher transfectionefficiency of 293T cells compared to Vero cells. These results show thatthe eight-plasmid system allows the generation of A/PR/8/34 (H1N1) fromcloned cDNA.

To compare the growth between the wildtype virus and the generatedrecombinant virus, embryonated hen's eggs were inoculated with wildtypevirus or recombinant virus. The allantoic fluid was harvested 48 hoursafter infection. The virus yield was determined by HA-assay. Althoughthe HA-titers differed between individual eggs, we found that bothviruses had HA-titers between 5120 and 10240 hemagglutination units,indicating that both viruses are high yielding isolates. Thus, therecombinant virus that was generated by DNA transfection has the samerobust culture phenotype as the parental isolate.

Discussion

The eight-plasmid system of the invention avoids the use of separateplasmids for protein expression (see Background of the Invention), thussimplifying the method of generation of influenza A virus entirely fromcloned cDNA. The production of vaccines involves the generation of avirus that is used as virus seed for the production of a vaccine viruseither in eggs or in cell culture. Efficacy of a vaccination programdepends on selecting a subtype that matches the circulating pathogenicstrains closely to stimulate a high specific antibody titer in thevaccinated population, resulting in efficient protection. The sixA/PR/8/34 master plasmids (pHW191-PB2, pHW192-PB1, pHW193-PA, pHW195-NP,pHW197-M, and pHW198-NS) encoding the internal influenza A genes can nowbe used in cotransfection with plasmids encoding the glycoproteins HAand NA of a currently circulating strain. The ability to manipulate eachgene segment will also allow us to evaluate which gene segment(s) areimportant for high yield growth of the reassortant viruses in eggs aswell as in cell culture.

The fact that we were able to generate two laboratory influenza virusstrains (A/WSN/33 (H1N1) and A/PR/8/34 (H1N1)) and one field isolate(A/Teal/HK/W312/97 (H6N1)) by cotransfecting only eight plasmidssuggests that this system is applicable for the development of liveattenuated influenza vaccines. Live attenuated influenza virus vaccinesadministered intranasally induce local, mucosal, cell-mediated andhumoral immunity. Cold-adapted (ca) reassortant (CR) viruses containingthe six internal genes of live, attenuated influenza A/Ann Arbor/6/60(H2N2) and the haemagglutinin (HA) and neuraminidase (NA) ofcontemporary wild-type influenza viruses appear to be reliablyattenuated. This vaccine has been shown to be efficacious in childrenand young adults (Keitel & Piedra In Textbook of Influenza, Nicholson etal., eds. 1998, 373-390. However, it may be too attenuated to stimulatean ideal immune response in elderly people, the major group of the20,000 to 40,000 individuals in the USA dying each year as a result ofinfluenza infection. The contribution of each segment to the attenuatedphenotype is still not well defined (Keitel & Piedra, supra). Thisinformation can be acquired only by the sequential introduction ofspecific, defined attenuating mutations into a virus. Since a detailedanalysis requires the testing of a large number of manipulated viruses,the construction and transfection of only eight plasmids simplifies thistask and reduces the time and cost to achieve this goal.

Example 5 Unidirectional RNA Polymerase I-Polymerase II TranscriptionSystem for the Generation of Influenza A Virus from Eight Plasmids

The previously Examples describe a system for the generation ofinfluenza A virus by cotransfecting only eight plasmids from whichnegative-sense vRNA and positive-sense mRNA are expressed (this work wassubsequently published; see Hoffmann et al., 2000, Proceedings of theNational Academy of Sciences, USA 97, 6108-6113). This Example describesthe establishment of a different transcription system for the expressionof virus-like RNAs, allowing the intracellular synthesis of noncappedpositive-sense cRNA and 5′-capped mRNA from one template. Cotransfectionof eight RNA pol I-pol II tandem promoter plasmids containing the cDNAof A/WSN/33 (H1N1) resulted in the generation of infectious influenza Avirus, albeit with lower virus yield than the bidirectional system. Ourapproach of producing either vRNA and mRNA or cRNA and mRNAintracellularly from a minimum set of plasmids is useful for theestablishment or optimization of reverse genetics systems of other RNAviruses.

The results reported in this Example were published (see Hoffmann andWebster, J. Gen. Virol. 2000, 81:2843).

For the generation of negative-sense RNA virus, either negative-sensevRNA or positive-sense cRNA can serve as a template. To reduce thenumber of plasmids needed for the recovery of virus, we reasoned that itmight be possible for cellular RNA pol I and pol II to synthesize cRNAand mRNA from one template. Therefore we attempted to develop aunidirectional pol I-pol II transcription system (FIG. 5). Viral cDNA isinserted in the positive-sense orientation between an RNA pol I promoterand a terminator sequence. This whole pol I transcription unit isinserted in the positive-sense orientation between an RNA pol IIpromoter and a polyadenylation site (FIG. 5). Unlike, the negative-sensevRNA and positive-sense mRNA generated in our bidirectionaltranscription system (FIG. 1), two types of positive-sense RNAs wereexpected to be synthesized. From the pol II promoter, an mRNA with a5′-cap structure should be transcribed in the nucleoplasm. Thistranscript should be translated into protein. In the nucleolus, cellularpol I is expected to synthesize full-length, positive-sense influenzavirus cRNA with a triphosphate group at the 5′ end (FIG. 5). A cloningvector, pHW11, that can be used for insertion of arbitrary cDNAfragments was constructed (FIG. 6). This plasmid contains the pol IIpromoter (immediate early promoter of the human cytomegalovirus) and thehuman pol I promoter that are upstream of a pol I terminator sequenceand a poly(A) site.

To test whether infectious influenza A virus can be generated bysynthesizing cRNA and mRNA from a single template, we constructed eightplasmids. The plasmids pHW171-PB2, pHW172-PB1, pHW173-PA, pHW174-HA,pHW175-NP, pHW176-NA, pHW177-M, and pHW178-NS contain the cDNAsrepresenting the eight gene segments of influenza A strain A/WSN/33(H1N1). All of these cDNAs are in the positive-sense orientation withregard to the pol I and pol II promoters. The eight plasmids (1 μg ofeach plasmid) were transfected into 293T or COS-1 cells with or withoutco-culturing with MDCK cells as described in Example 2.

The virus yield in the supernatant of transfected cells at differenttimes was determined by plaque assay after passage on MDCK cells.Forty-eight hours after transfection 2-5×10³ infectious virions wereproduced (Table 3). Seventy two hours after transfection the supernatantcontained 4×10⁴ pfu/ml after transfection of 293T or 2×10⁴ pfu/ml aftertransfection of COS-1 cells. The virus yield after 72 h could beincreased by co-culturing 293T cells or COS-1 cells with MDCK cells(Table 3).

The generation of virus proves that after transfection of the eightplasmids, RNA pol I synthesized the eight noncapped, positive-sensecRNAs. The four viral polymerase proteins translated from cellular RNApol II-synthesized transcripts bound to the naked virus-like cRNAs toform cRNPs. The polymerase subunit PB1 is important for the recognitionof the terminal structure and binding of the virus-like cRNAs (González& Ortín EMBO J. 1999, 18:3767; González & Ortín, J. Virol. 1999, 73:631;and 1999b; Li et al., EMBO J. 1998, 17:5844). The interaction with otherpolymerase proteins started the replication-transcription cycle, whichresulted in the synthesis of vRNPs and viral mRNAs (Toyoda et al., J.Gen. Virol. 1996, 77:2149; González et al., Nucl. Acids Res. 1996,29:4456). In the pol I-pol II transcription system, two different mRNAtypes are synthesized. One is directly transcribed from the plasmid-DNAby RNA pol II and contains the 225-nt pol I promoter sequence in the 5′end and the pol I terminator sequence in the 3′ end. Another mRNA issynthesized by viral polymerase complex proteins that use the vRNA astemplate. The 5′ cap structure of this mRNA is acquired by thecap-snatching mechanism in which the polymerase subunit PB2 takes thecap from cellular RNAs (Ulmanen et al., Proc. Natl. Acad. Sci. USA 1981,21:3607). Although both mRNA types differ in their 5′ and 3′ noncodingregions, they contain the same open reading frames for all viralproteins. The translated structural proteins together with the vRNPsassemble to create infectious influenza A virus. TABLE 3 Plasmid setsused for the production of A/WSN/33 (H1N1). Plasmids* unidirectionalsystem bidirectional system Virus gene segment 1 pHW172-PB2 pHW171-PB2pHW171-PB2 pHW171-PB2 pHW181-PB2 pHW181-PB2 pHW181-PB2 pHW181-PB2 2pHW172-PB1 pHW172-PB1 pHW172-PB1 pHW172-PB1 pHW182-PB1 pHW182-PB1pHW182-PB1 pHW182-PB1 3 pHW173-PA pHW173-PA pHW173-PA pHW173-PApHW183-PA pHW183-PA pHW183-PA pHW183-PA 4 pHW174-HA pHW174-HA pHW174-HApHW174-HA pHW184-HA pHW184-HA pHW184-HA pHW184-HA 5 pHW175-NP pHW175-NPpHW175-NP pHW175-NP pHW185-NP pHW185-NP pHW185-NP pHW185-NP 6 pHW176-NApHW176-NA pHW176-NA pHW176-NA pHW186-NA pHW186-NA pHW186-NA pHW186-NA 7pHW177-M pHW177-M pHW177-M pHW177-M pHW187-M pHW187-M pHW187-M pHW187-M8 pHW178-NS pHW178-NS pHW178-NS pHW178-NS pHW188-NS pHW188-NS pHW188-NSpHW188-NS 293T COS-1 COS-1 293T 293T COS-1 COS-1 Transfected 293T +MDCK+MDCK +MDCK +MDCK cells^(#) Transcripts^(†) cRNA and mRNA vRNA and mRNAVirus titer (pfu/ml)^(§) t = 24 h 0 0 0 0 5 × 10² 4 × 10² 1 × 10³ 1 ×10³ t = 48 h 4 × 10³ 5 × 10³ 2 × 10³ 5 × 10³ 8 × 10⁶ 1 × 10⁷ 6 × 10⁶ 1 ×10⁷ t = 72 h 4 × 10⁴ 2 × 10⁵ 2 × 10⁴ 4 × 10⁵ 1 × 10⁷ 2 × 10⁸ 1 × 10⁷ 3 ×10⁸*The plasmids with the unidirectional transcription units and theplasmids with bidirectional transcription units (FIG. 1) contain cDNAsrepresenting the eight gene segments of A/WSN/33 (H1N1).^(#)293T or COS-1 cells were transfected either without or withco-cultured MDCK cells.^(†)RNA transcripts synthesized by pol I or pol II.^(§)Virus titer of the supernatant was determined at the indicated times(24 h, 48 h, 72 h) after transfection by plaque assay on MDCK cells.

Although the generation of WSN-virus from cells transfected with eighttandem-promoter plasmids proved to be very reliable, the virus yield bythis cRNA-mRNA approach was lower than that of the bidirectional systemthat produces vRNA and mRNA transcripts (Table 3). Seventy-two hoursafter 293T or COS-1 cells had been transfected with the eight plasmidscontaining the bidirectional pol I-pol II transcription system (FIG. 1;Example 2; see Hoffmann et al., Proc. Nati. Acad. Sci. USA 2000,97:6108; pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA, pHW185-NP,pHW186-NA, pHW187-M, and pHW188-NS), the virus titer was 1×10⁷ pfu/ml(Table 3). Twenty four hours after transfection of COS-1 or 293T cells0.4-1×10³ pfu/ml were found in the supernatant. These data show that theeight plasmid bidirectional system has the same efficiency for virusgeneration with similar kinetics as the more complicated and cumbersomemulti plasmid system requiring cotransfection of 12 or 17 plasmids(Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345).

No infectious virus was found 24 h posttransfection with eight tandempromoter plasmids (Table 3). These results suggest that the differencesin virus yields between the vRNA-mRNA and cRNA-mRNA approaches are dueto the different polarities of the primary pol I transcripts. Thebidirectional system starts with the intracellular synthesis of vRNA, asituation resembling the natural influenza A infection in which vRNPsare transported to the nucleus and vRNAs initially serve as templatesfor mRNA and cRNA synthesis. In the unidirectional system, cRNPs are thefirst replication-competent units that are produced. To produce mRNAs,the cRNAs have to be replicated into vRNAs, and the vRNPs are ultimatelypackaged into progeny virus particles (Hsu et al., J. Gen. Virol. 1987,77:2575). Because of the additional reactions required for thegeneration of vRNPs from cRNPs, the formation of virus in theunidirectional system occurs at a later time than does virus formationby the bidirectional system.

Other possible reasons for the differences in virus yields of the twosystems are that sequence elements in the cDNA decrease the efficiencyof transcription by terminating transcription, or sequences in the RNAtranscripts reduce the steady-state level of the pol I or pol IItranscripts. A lower concentration of only one of the eight virus-likecRNAs or mRNAs reduces the overall efficiency of this system because allvRNPs and structural proteins have to be synthesized in concentrationsthat are optimal for virus replication and virus assembly.

The high efficiency of the eight-plasmid system for the generation ofinfluenza A virus indicates that this system applies to otherorthomyxoviruses, e.g., influenza B virus, influenza C virus, andThogotovirus. The results in this study suggest that the vRNA-mRNAsystem will be the most efficient way for generating these virusesentirely from plasmids. The present invention permits establishment ofpol I based systems for the generation of RNA viruses other than membersof the family Orthomyxoviridae, e.g., members of Paramyxoviridae,Arenaviridae or Bunyaviridae (Roberts, A. & Rose, J. K., Virology 1998,247:1-6; Bridgen & Elliot, Proc. Natl. Acad. Sci. USA 1996, 93:15400;Lee et al., J. Virol. 2000, 74:3470). Unlike orthomyxoviruses, most RNAviruses replicate in the cytoplasm of infected cells. During theirevolution the RNAs of these viruses have not been subjected to selectionpressures found in the nucleus, e.g. splicing. Generally, reversegenetics systems for nonsegmented negative strand RNA viruses are basedon the intracellular transcription from a T7 promoter as pioneered byConzelmann and colleages for the rescue of rabiesvirus (Schnell et al.,EMBO J. 1994, 13:4195). The expression of virus-like RNAs is driven byT7 RNA polymerase provided either by infection with a recombinantvaccinia virus or by using cell lines constitutively expressing T7 RNApolymerase. Unlike pol I transcription which, occurs in the nucleus,transcription by T7 RNA polymerase takes place in the cytoplasm. Use ofthe pol I transcription system for cytoplasmic RNA viruses would requirethat the RNA transcripts have to be transported out of the nucleus. Thatindeed pol I transcripts are transported out of the nucleus is supportedby the detection of protein production in cells containing pol Itranscripts that had an internal ribosomal entry site inserted into its5′ noncoding region (Palmer et al., Nucl. Acids. Res. 1993, 21:3451).Because information is limited about the sequences crucial for export orretention of pol I transcripts, synthesis of negative-sense orpositive-sense RNAs may result in different efficiencies of nuclearexport. In addition, the export of a large pol II-generatedcoronavirus-like transcript (having greater than 30,000 nts) from thenucleus (Almazán et al., Proc. Natl. Acad Sci. USA 2000, 97:5516)indicates that specific RNA sequences rather than the length of atranscript may be crucial for export. The pol I-pol II cloning vectorsthat we have developed and the efficient cloning method based on the useof type IIs restriction endonucleases will allow positive andnegative-sense RNA synthesized in the nucleus for the generation ofcytoplasmic RNA viruses at reasonable costs and within a reasonableperiod of time.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

All patents, patent applications, publications, and other materialscited herein are hereby incorporated herein reference in theirentireties.

1. An expression plasmid comprising an RNA polymerase I (pol I) promoterand pol I terminator sequences, which are inserted between an RNApolymerase II (pol II) promoter and a polyadenylation signal.
 2. Theexpression plasmid of claim 1 wherein the pol I promoter is proximal tothe polyadenylation signal and the pol I terminator sequence is proximalto the pol II promoter.
 3. The expression plasmid of claim 1 wherein thepol I promoter is proximal to the pol II promoter and the pol Iterminator sequence is proximal to the polyadenylation signal.
 4. Theexpression plasmid of claim 1 wherein the plasmid corresponds to aplasmid having a map selected from the group consisting of pHW2000,pHW11 and pHW12.
 5. The expression plasmid of claim 1, furthercomprising a negative strand RNA virus viral gene segment insertedbetween the pol I promoter and the termination signal.
 6. The expressionplasmid of claim 5, wherein the negative strand RNA virus is a member ofthe Orthomyxoviridae virus family.
 7. The expression plasmid of claim 6,wherein the virus is an influenza A virus.
 8. The expression plasmid ofclaim 7, wherein the viral gene segment encodes a gene selected from thegroup consisting of a viral polymerase complex protein, M protein, andNS protein; wherein the genes are derived from a strain well adapted togrow in cell culture or from an attenuated strain, or both.
 9. Theexpression plasmid of claim 6, wherein the virus is an influenza Bvirus.
 10. The expression plasmid of claim 8 wherein the plasmid has amap selected from the group consisting of pHW241-PB2, pHW242-PB1,pHW243-PA, pHW245-NP, pHW247-M, and pHW248-NS.
 11. The expressionplasmid of claim 8 wherein the plasmid has a map selected from the groupconsisting of pHW181-PB2, pHW182-PB1, pHW183-PA, pHW185-NP, pHW187-M,and pHW188-NS.
 12. The expression plasmid of claim 7, wherein the viralgene segment encodes a gene selected from the group consisting of aninfluenza hemagglutinin (HA) gene and a neuraminidase (NA) gene.
 13. Theexpression plasmid of claim 12, wherein the influenza gene is from apathogenic influenza virus strain.
 14. The expression plasmid of claim12, wherein the plasmid has a map selected from the group consisting ofpHW244-HA, pHW246-NA, pHW 184-HA, and pHW186-NA.
 15. A minimumplasmid-based system for the generation of infectious negative strandRNA viruses from cloned viral cDNA comprising a set of plasmids whereineach plasmid comprises one autonomous viral genomic segment, and whereinthe viral cDNA corresponding to the autonomous viral genomic segment isinserted between an RNA polymerase I (pol I) promoter and terminatorsequences, thereby resulting in expression of vRNA, which are in turninserted between a RNA polymerase II (pol II) promoter and apolyadenylation signal, thereby resulting in expression of viral mRNA.16. The minimum plasmid-based system of claim 15 wherein the pol Ipromoter is proximal to the polyadenylation signal and the pol Iterminator sequence is proximal to the pol II promoter.
 17. The minimumplasmid-based system of claim 15 wherein the pol I promoter is proximalto the pol II promoter and the pol I terminator sequence is proximal tothe polyadenylation signal.
 18. The plasmid-based system of claim 15,wherein the negative strand RNA virus is a member of theOrthomyxoviridae virus family.
 19. The plasmid-based system of claim 18,wherein the virus is an influenza A virus.
 20. The plasmid-based systemof claim 18, wherein the virus is an influenza B virus.
 21. Theplasmid-based system of claim 19, wherein the viral gene segment encodesa protein selected from the group consisting of a viral polymerasecomplex protein, an M protein and an NS protein; wherein said genes arefrom a strain well adapted to grow in cell culture or from an attenuatedstrain, or both.
 22. The plasmid-based system of claim 19, wherein theviral genomic segments comprise genes which encode a protein selectedfrom the group consisting of hemagglutinin and neuraminidase, or both;wherein said genes are from a pathogenic influenza virus.
 23. Theplasmid-based system of claim 19 wherein said system comprises one ormore plasmids having a map selected from the group consisting ofpHW241-PB2, pHW242-PB1, pHW243-PA, pHW244-HA, pHW245-NP, pHW246-NA,pHW247-M, and pHW248-NS.
 24. The plasmid-based system of claim 19,wherein said system comprises one or more plasmids having a map selectedfrom the group consisting of pHW181-PB2, pHW182-PB1, pHW183-PA,pHW184-HA, pHW185-NP, pHW186-NA, pHW187-M, and pHW188-NS.
 25. A hostcell comprising the plasmid-based system of claim
 15. 26. A host cellcomprising the plasmid-based system of claim
 18. 27. A host cellcomprising the plasmid-based system of claim
 19. 28. A host cellcomprising the plasmid-based system of claim
 22. 29. A method forproducing a negative strand RNA virus virion, which method comprisesculturing the host cell of claim 25 under conditions that permitproduction of viral proteins and vRNA or cRNA.
 30. A method forproducing an Orthomyxoviridae virion, which method comprises culturingthe host cell of claim 26 under conditions that permit production ofviral proteins and vRNA or cRNA.
 31. A method for producing an influenzavirion, which method comprises culturing the host cell of claim 27 underconditions that permit production of viral proteins and vRNA or cRNA.32. A method for producing a pathogenic influenza virion, which methodcomprises culturing the host cell of claim 28 under conditions thatpermit production of viral proteins and vRNA or cRNA.
 33. A method forpreparing a negative strand RNA virus-specific vaccine, which methodcomprises purifying a virion produced by the method of claim
 29. 34. Themethod according to claim 33, which further comprises inactivating thevirion.
 35. The method according to claim 33, wherein the negativestrand RNA virus is an attenuated virus.
 36. A method for vaccinating asubject against a negative strand RNA virus infection, which methodcomprises administering a protective dose of a vaccine of claim 33 tothe subject.
 37. A method for vaccinating a subject against a negativestrand RNA virus infection, which method comprises injecting aprotective dose of a vaccine of claim 33 intramuscularly in the subject.38. A method for vaccinating a subject against a negative strand RNAvirus infection, which method comprises administering a vaccine of claim33 intranasally to the subject.
 39. A method for generating anattenuated negative strand RNA virus, which method comprises: (a)mutating one or more viral genes in the plasmid-based system of claim15; and (b) determining whether infectious RNA viruses produced by thesystem are attenuated.
 40. A composition comprising a negative strandRNA virus virion, wherein viral internal proteins of the virion are froma virus strain well adapted to grow in culture or from an attenuatedstrain, or both and viral antigen proteins, of the virion are from apathogenic virus strain.
 41. A composition comprising a negative strandRNA virus virion produced by the method of claim 29.